Small cell lung cancer organoid culture medium and application method thereof

Abstract

The invention provides a small cell lung cancer organoid culture medium and an application method thereof. The small cell lung cancer organoid culture medium is prepared from the following components: 10 to 500 [mu] g / mL of R-spondin-1, 10 to 500 [mu] g / mL of Noggin, 0.01 to 0.2 mg / mL of EGF (Epidermal Growth Factor), 0.05 to 0.2 [mu] g / mL of FGFb (Fibroblast Growth Factor b), 0.1 to 10 [mu] g / mL of FGF10, 0.02 to 0.5 mM of A83-01, 0.5 to 20 mM of Y-27632, 1 to 500 mM of SB202190, 0.1 to 200 mM of Nicotinamide, 0.1 to 500 mM of Insulin, 0.1 to 50 nM of protodioscin, 1 to 100 [mu] All the components are dissolved in a DMEM / F12 culture solution. The small cell lung cancer organoid culture medium provided by the invention is suitable for small cell lung cancer tissue organoid culture after component concentration optimization design, good in stability, high in growth speed and remarkably improved in passage frequency, and can still maintain the same tissue structure and genome characteristics of primary tissues after multiple passages; and an ideal model can be provided for research on the occurrence and development mechanism of small cell lung cancer, detection and research on drug sensitivity and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of organoid culture, and in particular to a small cell lung cancer organoid culture medium and an application method thereof. Background Art

[0002] Small cell lung cancer (SCLC) is a high-grade neuroendocrine cancer that arises from heterogeneous neuroendocrine tumors of Kuchitsky cells in the bronchial mucosal epithelium. It is the most aggressive subtype of lung cancer, primarily occurring in current or former smokers and carrying a particularly poor prognosis. SCLC accounts for approximately 15-20% of lung cancer cases. SCLC patients typically present with respiratory symptoms, including cough, dyspnea (labored breathing), or hemoptysis (coughing up blood). Imaging studies reveal a centrally located lung mass, often with large thoracic lymph node involvement. Two-thirds of patients have developed distant metastases at the time of initial diagnosis. Current treatments for SCLC primarily include surgery, chemotherapy, radiotherapy, and immunotherapy and targeted therapies that have not yet been formally approved for clinical use. Drug-adjuvant therapy suffers from diverse regimens, low drug efficiency, the development of drug resistance, and complex drug testing procedures.

[0003] Organoids are organ-specific cell collections derived from stem cells or precursor cells. Organoids cultured in vitro are highly similar to their corresponding organs in terms of cellular composition and tissue architecture, and possess corresponding functional characteristics. Unlike conventional cell culture, which cultivates a single cell population in a two-dimensional environment, organoid culture cultivates a variety of cell populations contained in specific tissues and organs in a three-dimensional environment, and its culture system is more similar to the in vivo microenvironment. Therefore, it shows great application prospects in basic research on the physiology and pathology of various organs, precision medicine, drug screening and development, gene therapy, regenerative medicine, etc.

[0004] Chinese patent CN117025537A "A special culture medium for small cell lung cancer organoids and its application" discloses a special culture medium for small cell lung cancer organoids, which promotes the development of small cell lung cancer and helps to construct small cell lung cancer organoids. However, the culture medium has poor stability and is prone to contamination, which is not conducive to the long-term subculture of small cell lung cancer organoid cells.

[0005] Therefore, it is of great significance to provide a small cell lung cancer organoid culture medium with good stability and conducive to long-term subculture of small cell lung cancer and an application method thereof. Summary of the Invention

[0006] In view of the problems that the existing small cell lung cancer organoid culture medium has poor culture medium stability, is prone to contamination, and is not conducive to the long-term subculture of small cell lung cancer organoid cells, the present invention provides a small cell lung cancer organoid culture medium and its application method. After the component concentration is optimized and designed, it is suitable for the culture of small cell lung cancer tissue organoids, has good stability, fast growth rate, significantly increased number of subcultures, and can still maintain the same tissue structure and genomic characteristics of the original tissue after multiple subcultures.

[0007] To achieve the above object, the technical solution adopted by the present invention is: A small cell lung cancer organoid culture medium, comprising, according to final concentrations: 10-500 μg / mL R-spondin-1, 10-500 μg / mL Noggin, 0.01-0.2 mg / mL EGF, 0.05-0.2 μg / mL FGFb, 0.1-10 μg / mL FGF10, 0.02- 0.5 mM A83-01, 0.5-20 mM Y-27632, 1-500 mM SB202190, 0.1-200 mM Nicotinamide, 0.1-500 mM Insulin, 0.1-50 nM Protodioscin, 1-100 μM Dehydroepiandrosterone acetate, 0.5-50 ng / mL SCF, 20-500 μg / mL primary cell antibiotics; all the above ingredients are dissolved in DMEM / F12 culture medium.

[0008] Furthermore, the final concentration composition includes: 10-200 μg / mL R-spondin-1, 10-200 μg / mL Noggin, 0.01-0.1 mg / mL EGF, 0.05-0.1 μg / mL FGFb, 0.1-10 μg / mL FGF10, 0.02- 0.2 mM A83-01, 0.5-10 mM Y-27632, 1-200 mM SB202190, 0.1-100 mM Nicotinamide, 0.1-100 mM Insulin, 1-30 nM Protodioscin, 1-50 μM Dehydroepiandrosteroneacetate, 1-25 ng / mL SCF, 20-300 μg / mL primary cell antibiotics; all the above ingredients are dissolved in DMEM / F12 culture medium.

[0009] Furthermore, the preparation method is to dissolve the components in DMEM / F12 culture medium, mix them evenly, and form a liquid culture medium.

[0010] Another object of the present invention is to provide a method for using a small cell lung cancer organoid culture medium.

[0011] A method for culturing small cell lung cancer organoids, using any of the small cell lung cancer organoid culture media described above.

[0012] Further, the following steps are included: S1. Pre-treat the obtained small cell lung cancer specimen to obtain cell clusters of 3-50 cells; S2. Mix the cell pellet with the glue solution and plate it. After solidification, invert and fix it for 20-30 minutes. S3. Add small cell lung cancer organoid culture medium and culture for 4-7 days, during which the culture medium is replaced every 2-3 days to obtain the small cell lung cancer organoid.

[0013] Furthermore, the pretreatment in S1 includes: washing the obtained small cell lung cancer specimens with sterile saline, cutting them into pieces of 1-3 mm 3 Add 5 mL of type III collagenase and shake at 37°C for 30 min. After digestion, add 5 mL of DMEM / F12 medium to terminate digestion, centrifuge, and remove the supernatant.

[0014] Furthermore, the glue solution in S2 is Matrigel matrix glue.

[0015] Furthermore, the culture conditions in S3 are 37° C. and 5% CO 2 .

[0016] Another object of the present invention is to provide a small cell lung cancer organoid.

[0017] A small cell lung cancer organoid, characterized in that it is cultured according to any of the above-mentioned small cell lung cancer organoid culture methods.

[0018] Another object of the present invention is to provide an application of small cell lung cancer organoids.

[0019] The use of the above-mentioned small cell lung cancer organoids in screening drugs for preventing or treating small cell lung cancer.

[0020] Compared with the prior art, the present invention has the following advantages and beneficial effects: The small cell lung cancer organoid culture medium provided by the present invention contains the components required for the culture of small cell lung cancer tissue organoids, has a wide range of applications, and can culture tissues including squamous cell carcinoma and adenocarcinoma of the lung and normal lung. During the culture process, organoids are formed that are similar to small cell lung cancer in terms of cell composition and spatial structure. In addition, the small cell lung cancer organoid culture medium of the present invention does not require bovine serum albumin (FBS), the most common component in cell culture. The component composition is simple, which saves costs while reducing the cytotoxicity and inhibitors brought by FBS. The added specific cytokines (protodioscin, dehydroepiandrosterone acetate and SCF) are conducive to the rapid expansion of small cell lung cancer stem cells and the maturation and differentiation of organoids; the added inhibitors are all water-soluble inhibitors and do not require DMSO dissolution, which reduces the toxicity of DMSO to in vitro cell culture. The various components work synergistically to promote stem cell self-renewal, which is conducive to long-term subculture of small cell lung cancer. After the component concentration is optimized, it is suitable for the culture of small cell lung cancer tissue organoids, with a fast growth rate, a significantly increased number of subcultures, and the same tissue structure of the original tissue can be maintained after multiple subcultures. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The invention is further described with reference to the accompanying drawings, but the embodiments in the accompanying drawings do not constitute any limitation to the invention. A person skilled in the art can obtain other drawings based on the following drawings without making any creative effort.

[0022] Figure 1 This is an optical microscopic image of the small cell lung cancer organoid cultured in Example 4 of the present invention.

[0023] Figure 2 This is an optical microscopic image of the small cell lung cancer organoid cultured in Example 5 of the present invention.

[0024] Figure 3 This is an optical microscopic image of the small cell lung cancer organoid cultured in Example 6 of the present invention.

[0025] Figure 4 This is an optical microscope image of the small cell lung cancer organoids cultured in Comparative Example 1 of the present invention.

[0026] Figure 5 This is an optical microscope image of the small cell lung cancer organoids cultured in Comparative Example 2 of the present invention.

[0027] Figure 6 This is an optical microscope image of the small cell lung cancer organoids cultured in Comparative Example 3 of the present invention.

[0028] Figure 7 This is an optical microscope image of the small cell lung cancer organoids cultured in Comparative Example 4 of the present invention.

[0029] Figure 8 This is an optical microscope image of the small cell lung cancer organoids cultured in Comparative Example 5 of the present invention.

[0030] Figure 9 This is an optical microscopic image of the small cell lung cancer organoid cultured in Example 6 at the second generation.

[0031] Figure 10 This is an optical micrograph of the small cell lung cancer organoid cultured in Example 6 at the fifth generation. DETAILED DESCRIPTION

[0032] To better illustrate the objectives, technical solutions, and advantages of the present invention, the present invention is further described by the following examples. Obviously, the following examples are only a part of the embodiments of the present invention, rather than all the embodiments; it should be understood that the embodiments of the present invention are only used to illustrate the technical effects of the present invention, and are not used to limit the scope of protection of the present invention.

[0033] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in this specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0034] Example 1: A small cell lung cancer organoid culture medium, comprising, at final concentrations, 50 μg / mL R-spondin-1, 20 μg / mL Noggin, 0.05 mg / mL EGF, 0.1 μg / mL FGFb, 5 μg / mL FGF10, 0.1 mM A83-01, 5 mM Y-27632, 100 mM SB202190, 50 mM Nicotinamide, 50 mM Insulin, 30 nM Protodioscin, 50 μM Dehydroepiandrosterone acetate, 20 ng / mL SCF, and 200 μg / mL primary cell antibiotics; the components are dissolved in DMEM / F12 culture medium and mixed evenly to form a liquid culture medium, thereby obtaining the small cell lung cancer organoid culture medium.

[0035] Example 2: A small cell lung cancer organoid culture medium, comprising, at final concentrations, 80 μg / mL R-spondin-1, 10 μg / mL Noggin, 0.1 mg / mL EGF, 0.5 μg / mL FGFb, 8 μg / mL FGF10, 0.05 mM A83-01, 10 mM Y-27632, 50 mM SB202190, 100 mM Nicotinamide, 100 mM Insulin, 20 nM Protodioscin, 20 μM Dehydroepiandrosterone acetate, 10 ng / mL SCF, and 200 μg / mL primary cell antibiotics; the components are dissolved in DMEM / F12 culture medium and mixed evenly to form a liquid culture medium, thereby obtaining the small cell lung cancer organoid culture medium.

[0036] Example 3: A small cell lung cancer organoid culture medium, which comprises, in final concentration, 50 μg / mL R-spondin-1, 20 μg / mL Noggin, 0.5 mg / mL EGF, 0.1 μg / mL FGFb, 10 μg / mL FGF10, 0.01 mM A83-01, 1 mM Y-27632, 100 mM SB202190, 100 mM Nicotinamide, 50 mM Insulin, 10 nM Protodioscin, 10 μM Dehydroepiandrosterone acetate, 5 ng / mL SCF, and 100 μg / mL primary cell antibiotic; the components are dissolved in DMEM / F12 culture medium and mixed evenly to form a liquid culture medium, thereby obtaining the small cell lung cancer organoid culture medium.

[0037] Example 4: A method for culturing small cell lung cancer organoids, comprising the following steps: S1. Transfer the obtained small cell lung cancer specimen to a centrifuge tube, then wash with sterile saline for 30 seconds, remove the supernatant, and re-add sterile saline for washing. Repeat the above washing method three times to remove impurities on the tissue surface. In a biosafety cabinet, transfer the sample to a 6 cm culture dish and mince the tissue into 1-3 mm pieces using sterilized surgical scissors on ice. 3 , add 5 mL of type III collagenase and shake at 37°C for 30 min. After digestion, add 5 mL of DMEM / F12 medium to terminate digestion, centrifuge at 1000 rpm for 5 min, and carefully remove the supernatant to obtain cell pellets with 3-50 cells; S2. Mix the cell pellet with Matrigel and plate it at a 1:1 ratio. After solidification for 2 minutes, invert and incubate for 20 minutes. S3. Add the small cell lung cancer organoid culture medium obtained in Example 1, and then place in a constant temperature incubator at 37°C and 5% CO2 concentration for 5 days, during which the culture medium is replaced every 2 days to obtain the small cell lung cancer organoid.

[0038] Example 5: A method for culturing small cell lung cancer organoids, comprising the following steps: S1. Transfer the obtained small cell lung cancer specimen to a centrifuge tube, then wash with sterile saline for 30 seconds, remove the supernatant, and re-add sterile saline for washing. Repeat the above washing method three times to remove impurities on the tissue surface. In a biosafety cabinet, transfer the sample to a 6 cm culture dish and mince the tissue into 1-3 mm pieces using sterilized surgical scissors on ice. 3 , add 5 mL of type III collagenase and shake at 37°C for 30 min. After digestion, add 5 mL of DMEM / F12 medium to terminate digestion, centrifuge at 1000 rpm for 5 min, and carefully remove the supernatant to obtain cell pellets with 3-50 cells; S2. Mix the cell pellet with Matrigel and plate it at a ratio of 1:1.5. After solidification for 5 minutes, invert and incubate for 30 minutes. S3. Add the small cell lung cancer organoid culture medium obtained in Example 2, and then place in a constant temperature incubator at 37°C and 5% CO2 concentration for 5 days, during which the culture medium is replaced every 2 days to obtain the small cell lung cancer organoid.

[0039] Example 6: A method for culturing small cell lung cancer organoids, comprising the following steps: S1. Transfer the obtained small cell lung cancer specimen to a centrifuge tube, then wash with sterile saline for 30 seconds, remove the supernatant, and re-add sterile saline for washing. Repeat the above washing method three times to remove impurities on the tissue surface. In a biosafety cabinet, transfer the sample to a 6 cm culture dish and mince the tissue into 1-3 mm pieces using sterilized surgical scissors on ice. 3 , add 5 mL of type III collagenase and shake at 37°C for 30 min. After digestion, add 5 mL of DMEM / F12 medium to terminate digestion, centrifuge at 1000 rpm for 5 min, and carefully remove the supernatant to obtain cell pellets with 3-50 cells; S2. Mix the cell pellet with Matrigel and plate it at a ratio of 1:1.3. After solidification for 3 minutes, invert and incubate for 30 minutes. S3. Add the small cell lung cancer organoid culture medium obtained in Example 3, and then place in a constant temperature incubator at 37°C and 5% CO2 concentration for 5 days, during which the culture medium is replaced every 2 days to obtain the small cell lung cancer organoid.

[0040] Comparative Example 1: A culture medium for small cell lung cancer organoids. The steps not otherwise specified are the same as those in Example 3, except that: Protodioscin was not added in this comparative example.

[0041] Small cell lung cancer organoids were obtained after culturing for 5 days using the culturing method of Example 6.

[0042] Comparative Example 2: A culture medium for small cell lung cancer organoids. The steps not otherwise specified are the same as those in Example 3, except that: In this comparative example, no dehydroepiandrosterone acetate was added.

[0043] Small cell lung cancer organoids were obtained after culturing for 5 days using the culturing method of Example 6.

[0044] Comparative Example 3: A culture medium for small cell lung cancer organoids. The steps not otherwise specified are the same as those in Example 3, except that: In this comparative example, no SCF was added.

[0045] Small cell lung cancer organoids were obtained after culturing for 5 days using the culturing method of Example 6.

[0046] Comparative Example 4: A culture medium for small cell lung cancer organoids. The steps not otherwise specified are the same as those in Example 3, except that: The final concentration of Protodioscin in this comparative example was 0.05 nM.

[0047] Small cell lung cancer organoids were obtained after culturing for 5 days using the culturing method of Example 6.

[0048] Comparative Example 5: A culture medium for small cell lung cancer organoids. The steps not otherwise specified are the same as those in Example 3, except that: The final concentration of Protodioscin in this comparative example was 100 nM.

[0049] Small cell lung cancer organoids were obtained after culturing for 5 days using the culturing method of Example 6.

[0050] The small cell lung cancer organoids obtained in Examples 4-6 and Comparative Examples 1-5 were observed under a microscope. The optical microscope images obtained are as follows: Figure 1-8 shown.

[0051] Figure 1 、 2 3 are optical microscopic images of the small cell lung cancer organoids cultured in Examples 4, 5, and 6 of the present invention. It can be observed that the small cell lung cancer organoid culture medium of the present invention can produce a large number of small cell lung cancer organoid clusters using a small amount of tumor tissue from small cell lung cancer patients, with stable morphology and uniform size.

[0052] Figure 4 、 5 , 6, 7, and 8 are optical micrographs of the small cell lung cancer organoids cultured in Comparative Examples 1, 2, 3, 4, and 5 of the present invention. It can be observed that when a specific component is missing or the final concentration exceeds the range, the cultured small cell lung cancer organoids are significantly smaller than those in Example 6, and the effect is poor.

[0053] Figure 9 、 10 Images of small cell lung cancer tissue organoids cultured in Example 6 at the second and fifth passages. It can be observed that the small cell lung cancer organoid culture medium of the present invention, after optimizing the concentration of its components, is suitable for culturing small cell lung cancer tissue organoids, significantly improving the number of passages and maintaining the same histological structure of the primary tissue after multiple passages, demonstrating excellent stability.

[0054] In summary, the small cell lung cancer organoid culture medium provided by the present invention contains the components required for the culture of small cell lung cancer tissue organoids, has a wide range of applications, and can culture tissues including lung squamous cell carcinoma, lung adenocarcinoma, and normal lung origin. The component composition is simple, and the added specific cytokines are conducive to the rapid expansion of small cell lung cancer stem cells and the maturation and differentiation of organoids; the synergistic effect of each component is conducive to the long-term subculture of small cell lung cancer. After the component concentration is optimized, it is suitable for the culture of small cell lung cancer tissue organoids, has a fast growth rate, and the number of subcultures is significantly increased. After multiple subcultures, the same tissue structure of the original tissue can still be maintained.

[0055] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A small cell lung cancer organoid culture medium, characterized in that The final concentration composition includes: 10-500 μg / mL R-spondin-1, 10-500 μg / mL Noggin, 0.01-0.2 mg / mL EGF, 0.05-0.2 μg / mL FGFb, 0.1-10 μg / mL FGF10, 0.02- 0.5 mM A83-01, 0.5-20 mM Y-27632, 1-500 mM SB202190, 0.1-200 mM Nicotinamide, 0.1-500 mM Insulin, 0.1-50 nM Protodioscin, 1-100 μM Dehydroepiandrosterone acetate, 0.5-50 ng / mL SCF, 20-500 μg / mL primary cell antibiotics; all of the above ingredients are dissolved in DMEM / F12 culture medium.

2. A small cell lung cancer organoid culture medium according to claim 1, characterized in that The final concentration composition includes: 10-200 μg / mL R-spondin-1, 10-200 μg / mL Noggin, 0.01-0.1 mg / mL EGF, 0.05-0.1 μg / mL FGFb, 0.1-10 μg / mL FGF10, 0.02- 0.2 mM A83-01, 0.5-10 mM Y-27632, 1-200 mM SB202190, 0.1-100 mM Nicotinamide, 0.1-100 mM Insulin, 1-30 nM Protodioscin, 1-50 μM Dehydroepiandrosterone acetate, 1-25 ng / mL SCF, 20-300 μg / mL primary cell antibiotics; all of the above ingredients are dissolved in DMEM / F12 culture medium.

3. A small cell lung cancer organoid culture medium according to claim 1 or 2, characterized in that: The preparation method comprises the following steps: dissolving the components in DMEM / F12 culture fluid, mixing the mixture evenly, and forming a liquid culture medium.

4. A method for culturing small cell lung cancer organoids, characterized in that: Use the small cell lung cancer organoid culture medium according to any one of claims 1 to 3.

5. A method for culturing small cell lung cancer organoids according to claim 4, characterized in that: The following steps are involved: S1. Pre-treating the obtained small cell lung cancer specimen to obtain a cell cluster with a cell number of 3-50 cells; S2. Mix the cell mass and the glue solution and spread them on a plate. After solidification, invert and fix for 20-30 minutes. S3. Add small cell lung cancer organoid culture medium and culture for 4-7 days, during which the culture medium is replaced every 2-3 days to obtain the small cell lung cancer organoid.

6. A method for culturing small cell lung cancer organoids according to claim 5, characterized in that: S1 The pretreatment includes: washing the obtained small cell lung cancer specimens with sterile saline, cutting them into pieces of 1-3 mm 3 Size, add 5 mL type III collagenase, shake and digest at 37℃ for 30 min. After digestion, add 5 mL DMEM / F12 medium to terminate digestion, centrifuge and remove the supernatant.

7. A method for culturing small cell lung cancer organoids according to claim 5, characterized in that: The glue solution S2 is Matrigel matrix glue.

8. A method for culturing small cell lung cancer organoids according to claim 5, characterized in that: The culture conditions in S3 are 37° C. and 5% CO 2 .

9. A small cell lung cancer organoid, characterized in that The small cell lung cancer organoid is obtained by culturing according to the culturing method of any one of claims 4 to 8.

10. Use of the small cell lung cancer organoid according to claim 9 in screening drugs for preventing or treating small cell lung cancer.

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