Human small cell lung cancer brain metastasis organoid culture medium and culture method

Abstract

The invention discloses a human small cell lung cancer brain metastasis organoid culture medium and a culture method. According to the culture medium, an advanced DMEM / F12 culture medium is used as a basic culture medium, and the following components are added: 2 to 5 [mu] M of GlutaMax, 8 to 10 [mu] M of HEPES, 1 * to 2 * of B27, 500 [mu] M of SB202190, 5 to 10 [mu] M of Y-27632, 0.5 [mu] M of A83-01, 0.5 to 2 [mu] M of N-Acetyl-L-cysteine, 5 mM of Nicotinamide, 25 to 100 ng / ml of recombinant human FGF7, 100 to 500 ng / ml of recombinant human FGF10, 100 to 200 ng / ml of Human Noggin, 50 to 500 ng / ml of Human R-Spondin-1, 50 to 100 ng / ml of According to the culture method, TryPLE Express is used for dissociation, the time is short, the activity of dissociated single cells is high, and the success rate of organoid culture can be increased to the maximum extent. Meanwhile, by combining with a culture medium with an optimized formula proportion, formation and growth of brain metastatic organs of the human small cell lung cancer can be promoted, and the construction success rate is increased.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology, and specifically relates to a culture medium and a culture method for human small cell lung cancer brain metastasis organoids. Background Art

[0002] Lung cancer is one of the leading causes of cancer-related deaths worldwide, with small cell lung cancer (SCLC) accounting for approximately 15% of lung cancer cases. SCLC is characterized by its highly invasive nature, rapid growth, and early propensity to metastasize to distant sites. Brain metastasis is one of the most common sites of distant metastasis, with an incidence as high as 40% to 60%. Once brain metastasis occurs, the patient's prognosis is extremely poor, with a significantly shortened survival period, placing a heavy burden on patients and their families. SCLC also exhibits significant inter- and intratumor heterogeneity, which is significantly associated with tumor evolution, metastasis, and acquired resistance to treatment. Therefore, in-depth research into the pathogenesis of brain metastasis in small cell lung cancer and the search for effective treatments are of extremely important clinical significance.

[0003] In recent years, organoid technology, as an emerging in vitro model construction technology, has received widespread attention and rapid development in the field of tumor research. Patient-derived organoids (PDOs) refer to tissue or cell samples obtained from patients, which are used to form organoid models with specific tissue or organ structures and functions through three-dimensional culture technology in vitro. These organoids can highly retain the histological, genetic, and biological characteristics of the patient's original tissue, including heterogeneity within and between tumors, and can better reflect the growth environment and biological behavior of tumor cells in vivo, providing a more accurate and reliable platform for studying the biological characteristics of tumors, drug screening, and personalized treatment.

[0004] At present, although there have been many research reports on lung cancer organoid culture media and modeling methods, there are still some problems and challenges in the cultivation of human small cell lung cancer brain metastasis organoids. On the one hand, the cultivation of small cell lung cancer brain metastasis organoids is more difficult, and its requirements for culture conditions and nutrients are more stringent. The existing culture medium components and formulas are insufficient in supporting the growth of small cell lung cancer brain metastasis organoids and maintaining their characteristics, which may lead to problems such as slow growth of organoids and susceptibility to necrosis. On the other hand, the existing modeling methods are not perfect in terms of tissue sample processing, cell digestion and optimization of culture conditions, making it difficult to efficiently obtain representative and stable organoid models. For example, the Chinese patent "A culture medium and modeling method for difficult-to-culture lung cancer brain metastasis organoids" (CN202311835378.9) discloses a culture medium and culture method for lung cancer brain metastasis organoids. The culture medium includes Advanced DMEM / F12 50%, Neurobasal™ 50%, and Glutama 1%; antibiotics include Primocin 50-200 μg / ml, Penicillin-Streptomycin 50-200 U / ml and 50-200 mg / ml, respectively; growth factors include R-Spondin 10-500 ng / ml, Noggin 10-500 ng / ml, EGF 10-100 ng / ml, FGF10 50-200 ng / ml, FGF7 10-50 ng / ml, and Insulin 1-5 μg / ml; small molecules include B27 0.5-2%, N-AcetylCysteine 0.5-2 mM, and Nicotinamide 1~20mM, A83-010.1~1μM, Y276321μM~20μM. The tissue digestion enzyme in this patent contains DMEM basal culture medium, 5~20% fetal bovine serum, 0.1~2mg / ml type IV collagenase, Y276321μM~20μM, Primocin 80~120μg / ml, 100U / ml penicillin / 100mg / ml streptomycin. The digestion time is 3~10 hours. First of all, the culture medium is complex in composition and expensive, which limits its popularity in experimental research. In addition, the sample digestion liquid has complex composition, long digestion time, and is not suitable for the culture of small cell lung cancer brain metastasis organoids. Using this culture medium to culture small cell lung cancer brain metastasis organoids not only grows slowly, but the formed organoids are prone to blackening and necrosis, and are in poor condition.

[0005] In summary, the present invention aims to develop a novel culture medium and culture method to meet the special needs of culturing and modeling human small cell lung cancer brain metastasis organoids, so as to overcome the shortcomings of existing technologies and provide more effective tools and means for the research and treatment of small cell lung cancer brain metastasis. Summary of the Invention

[0006] In view of this, the purpose of the present invention is to provide a culture medium and culture method for human small cell lung cancer brain metastasis organoids to promote the formation and growth of human small cell lung cancer brain metastasis organoids, effectively avoid necrosis and blackening of organoids during the culture process, and improve the success rate of constructing human small cell lung cancer brain metastasis organoids.

[0007] In order to achieve the above object, the present invention provides the following technical solutions:

[0008] The present invention provides a culture medium for human small cell lung cancer brain metastasis organoids. The culture medium is based on advanced DMEM / F12 culture medium and is supplemented with the following components: GlutaMax 2-5 mM, HEPES 8-10 mM, B271×-2×, SB202190 500 mM, Y-27632 5-10 μM, A83-01 0.5 μM, N-Acetyl-L-cysteine 0.5-2 mM, Nicotinamide 5 mM, recombinant human FGF7 25-100 ng / ml, recombinant human FGF10 100-500 ng / ml, Human Noggin 100-200 ng / ml, Human R-Spondin-1 50-500 ng / ml, MurineWnt-3a 50-100 ng / ml, FLI-062-10 μM, penicillin-streptomycin 1×, Primocin 5~10μM.

[0009] The culture medium provided by the present invention, the advanced DMEM / F-12 culture medium, contains ethanolamine, glutathione, ascorbic acid, insulin, transferrin, and various trace elements, and is suitable for the culture of stem cells, epithelial cells, etc. GlutaMax in the culture system can improve the stability of the culture medium and optimize cell metabolism; HEPES can maintain the stability of the pH value of the culture system, while maintaining the balance of the osmotic pressure of the culture medium to prevent cells from being damaged by changes in osmotic pressure; B27 is a serum-free culture medium additive that can provide a variety of nutrients required for cell growth, including antioxidants, proteins, vitamins, and fatty acids, which helps maintain the three-dimensional structure of organoids and the interaction between cells, so that they can better simulate the tissue structure and function of lung cancer in vivo; SB202190 is a selective p38 MAPK inhibitor that inhibits p38 Y-27632 is a Rho kinase inhibitor that can reduce apoptosis caused by cell separation by inhibiting the activity of Rho kinase; A83-01 reduces cell death caused by excessive contraction by inhibiting the TGF-β signaling pathway; NAC (N-Acetyl-L-cysteine) is a potent antioxidant that can provide sulfhydryl groups (-SH), which not only helps maintain the redox balance in cells, but also can effectively remove reactive oxygen species (ROS) in cells and reduce the damage to cells caused by oxidative stress; Nicotinamide is a precursor of NAD+ (nicotinamide adenine dinucleotide) that can increase the level of NAD+ in cells. NAD+ plays a key role in cellular energy metabolism and redox reactions. The addition of Nicotinamide helps maintain the metabolic homeostasis of cells and supports the normal growth and function of organoids. In addition, nicotineamide has antioxidant properties, scavenging intracellular reactive oxygen species (ROS) and reducing oxidative stress-induced cell damage. FGF7, a member of the fibroblast growth factor family, activates downstream signaling pathways, promotes the proliferation and differentiation of lung cancer cells, and induces the production of larger lung cancer organoids. It also plays a role in the maintenance and expansion of lung cancer stem cells, enabling them to better mimic the pathological characteristics of lung cancer. FGF10 can induce foregut spheroids to differentiate into lung organoids, promoting the formation of alveolar and airway-like structures. Noggin is a secreted protein that specifically binds to and inhibits the activity of members of the bone morphogenetic protein (BMP) family. By inhibiting the BMP signaling pathway, Noggin can promote Wnt / β-catenin signaling, maintain stem cell stemness and proliferation, and help maintain the three-dimensional structure and intercellular interactions of lung cancer organoids, enabling them to better mimic the tissue structure and function of lung cancer in vivo. R-Spondin-1 can interact with related proteins in the Wnt signaling pathway, enhancing Wnt signaling.In lung cancer organoid cultures, it activates the Wnt / β-catenin signaling pathway, promoting cell proliferation and differentiation, maintaining stem cell stemness, and helping to improve the stability and maturity of lung cancer organoids, enabling long-term in vitro culture and passage. Wnt-3a, a key ligand in the Wnt signaling pathway, binds to the Frizzled receptor on the cell surface, activating the Wnt / β-catenin signaling pathway. In lung cancer organoid cultures, it activates this signaling pathway, promoting cell proliferation, differentiation, and survival. During the induction of lung organoid formation, it promotes the formation of alveolar and airway-like structures. FLI-06 is an inhibitor of the Notch signaling pathway, a highly conserved intercellular signaling pathway that plays a key role in the development of lung neuroendocrine cells. FLI-06 can promote the proliferation of SCLC. These added ingredients not only improve cell survival and proliferation efficiency but also help maintain organoid stability and long-term proliferation.

[0010] The present invention also provides a method for culturing human small cell lung cancer brain metastasis organoids, comprising the following steps:

[0011] S1. Place the fresh tissue sample in 4°C pre-cooled tissue preservation solution and transport it to the laboratory on ice.

[0012] S2. Remove the tissue sample from the biosafety cabinet and wash it in pre-chilled DPBS.

[0013] S3. Place the tissue in a cell culture dish and trim the sample using sterile forceps and tissue scissors to remove necrotic tissue and encapsulation.

[0014] S4. Transfer the tissue to a pre-chilled EP tube and mince the tissue sample using sterile scissors.

[0015] S5. Transfer the tissue fragments to a centrifuge tube, add 1× TryPLE Express Dissociation Buffer, and place in a 37°C incubator for dissociation.

[0016] S6. Centrifuge at 4°C to remove dissociation buffer;

[0017] S7. Wash the pellet with DMEM / F12 and sieve to remove undissociated tissue.

[0018] S8. Centrifuge at 4°C to obtain single cell pellets and count them.

[0019] S9. Resuspend the pellet to 2000–5000 cells per μL in advanced DMEM / F-12 according to the cell count results. Resuspend the cell suspension in Matrigel at a 1:1 volume ratio and place on ice.

[0020] S10. Remove the preheated 24-well plate and inoculate the cell resuspension into the 24-well plate. Transfer the plate to a cell culture incubator. After the matrix gel solidifies, add the above-mentioned human small cell lung cancer brain metastasis organoid culture medium. Add PBS to the remaining wells to reduce evaporation. Culture at 37°C and 5% CO2. Change the medium every 2-3 days. After 5-7 days of passaging, the cells can be used for downstream experiments.

[0021] Preferably, the tissue preservation solution in S1 is based on advanced DMEM / F12 culture medium, supplemented with the following reagents: penicillin 60 mg / L, streptomycin 0.1 g / L, Y-27632 10 μM, stored at 4° C., and used freshly prepared.

[0022] Preferably, the number of DPBS washing times in S2 is 2 to 3 times.

[0023] Preferably, in S4 , the tissue sample is cut into pieces of about 1 to 3 mm using sterile scissors.

[0024] Preferably, the amount of 1×TryPLE Express added to S5 is 8 to 10 times the volume of the tissue fragments.

[0025] Preferably, in S5, the dissociation conditions are: the rotation speed is set to 60 rpm / min, and the dissociation time is 20 to 30 minutes.

[0026] Preferably, in S6, the conditions for centrifugation to remove the dissociation solution are 250×g and 5 min.

[0027] Preferably, in S7, when sieving to remove undissociated tissue, the tissue is sieved through a 100 μm sieve.

[0028] Preferably, in S8, the centrifugation conditions for obtaining a single cell pellet are 250×g and 5 min.

[0029] Preferably, in S10, the inoculation method is to vertically inoculate 50 ul / well to the center of the bottom of the 24-well plate without touching the side walls of the 24-well plate.

[0030] Preferably, in S10, the method for adding the human small cell lung cancer brain metastasis organoid culture medium is to slowly add 700 μl / Well along the side wall.

[0031] Preferably, in S10, the method of subculturing is as follows: remove the culture medium, add 1 ml of TrypLE Express to each well, gently pipette 5 to 10 times, transfer to a centrifuge tube rinsed with anti-adhesion solution, dissociate on a constant temperature shaker at 37°C for 15 minutes, centrifuge at 4°C, 250×g, 5 minutes, remove the supernatant, add DMEM / F12 for washing, centrifuge again and sieve, and perform subsequent operations as the primary culture, add matrix gel and culture on plates.

[0032] In some embodiments, the culturing method of the human small cell lung cancer brain metastasis organoids also includes freezing, and the freezing method is to select organoids with good growth conditions, remove the culture medium and add DPBS, gently blow with the tip of the gun to remove the mixture of organoids and matrix gel, transfer to a centrifuge tube and centrifuge, discard the supernatant and add pre-cooled freezing solution, blow and mix well, and then transfer to a freezing tube, 1 ml per tube, put it in a programmed cooling box, place it in a -80°C refrigerator, and transfer it to a liquid nitrogen tank for storage overnight.

[0033] In some embodiments, the culture method of the human small cell lung cancer brain metastasis organoid further includes resuscitation, and the resuscitation method is to preheat the human small cell lung cancer brain metastasis organoid culture medium and DMEM / F12 culture medium to 37°C in a water bath, take out the cryopreservation tube, quickly place it in a 37°C water bath to thaw it quickly, transfer it to 5-8 ml of DMEM / F12, and take 1 ml of DMEM / F12 again to clean the cryopreservation tube to reduce residue; after centrifugation, remove the supernatant to obtain the organoid precipitate.

[0034] Beneficial Effects: Compared with existing technologies, the present invention utilizes TryPLE Express for dissociation, resulting in shorter dissociation times and higher activity of dissociated single cells, maximizing the success rate of organoid culture. Furthermore, by optimizing the culture medium ratio, the present invention promotes the formation and growth of organoids derived from brain metastases of human small cell lung cancer, improving the success rate of their establishment.

[0035] The present invention uses TryPLE Express for dissociation to more efficiently obtain active small cell lung cancer brain metastasis cells from the patient's body, while minimizing cell damage during the dissociation process, ensuring high cell survival rate and good growth state in subsequent culture, and laying a solid foundation for the successful culture of organoids. In terms of culture methods, the culture medium formula used in the present invention has been carefully designed and optimized, and a variety of key ingredients have been added. These ingredients can provide sufficient nutritional support and a suitable growth environment for the growth of small cell lung cancer brain metastasis organoids. Experimental results show that the growth rate of small cell lung cancer brain metastasis organoids is significantly improved by using the culture method of the present invention. Compared with traditional methods, the volume growth rate of the organoids is accelerated and the cell proliferation ability is enhanced. More importantly, the culture method of the present invention can effectively avoid the occurrence of necrosis and blackening of organoids during the culture process. The improvement of this phenomenon not only reflects the healthy state of the cells inside the organoids, but also shows that the culture conditions of the present invention can better simulate the in vivo environment, reduce cell stress response and metabolic disorders, thereby maintaining the stability and functionality of the organoids. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1These are images of primary tissue cultured on the third day after dissociation, wherein A is an image of the comparative culture method; and B is an image of the culture method of Example 1 of the present invention.

[0037] Figure 2 These are images of primary tissue cultured on the 7th day after dissociation, where A is an image of the comparative culture method; and B is an image of the culture method of Example 1 of the present invention.

[0038] Figure 3 These are images of the P2 generation cultured on the 5th day using the method of the present invention, wherein A is a 4× image; B is a 10× image; C is a 20× image; and D is a 40× image.

[0039] Figure 4 These are images of P1 cells cultured using the method of the present invention on the fourth day after cryopreservation and thawing, wherein A is a 4× image; B is a 10× image; C is a 20× image; and D is a 40× image.

[0040] Among them, A-1, A-2, and A-3 represent three images collected at the same magnification and numbered respectively. B-1, B-2, and B-3; C-1, C-2, and C-3; and D-1, D-2, and D-3 are all the same. DETAILED DESCRIPTION

[0041] The technical solutions of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative of and explain the present invention, and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above contents of the present invention are encompassed within the scope that the present invention is intended to protect. It should be noted that the experimental materials whose sources are not indicated in the examples of the present invention can all be obtained commercially, and the experimental methods for which specific conditions are not indicated in the examples of the present invention are generally carried out in accordance with conventional experimental methods in the art or in accordance with the methods recommended by the experimental material manufacturers.

[0042] The present invention discloses a method for culturing human small cell lung cancer brain metastasis organoids, comprising the following steps:

[0043] S1. Place the fresh tissue sample in 4°C pre-cooled tissue preservation solution and transport it to the laboratory on ice.

[0044] S2. Remove the tissue sample from the biosafety cabinet and wash it in pre-chilled DPBS.

[0045] S3. Place the tissue in a cell culture dish and trim the sample using sterile forceps and tissue scissors to remove necrotic tissue and encapsulation.

[0046] S4. Transfer the tissue to a pre-chilled EP tube and mince the tissue sample using sterile scissors.

[0047] S5. Transfer the tissue fragments to a centrifuge tube, add 1× TryPLE Express Dissociation Buffer, and place in a 37°C incubator for dissociation.

[0048] S6. Centrifuge at 4°C to remove dissociation buffer;

[0049] S7. Wash the pellet with DMEM / F12 and sieve to remove undissociated tissue.

[0050] S8. Centrifuge at 4°C to obtain single cell pellets and count them.

[0051] S9. Resuspend the pellet to 2000–5000 cells per μL in advanced DMEM / F-12 according to the cell count results. Resuspend the cell suspension in Matrigel at a 1:1 volume ratio and place on ice.

[0052] S10. Remove the preheated 24-well plate, inoculate the cell resuspension into the 24-well plate, transfer the plate to a cell culture incubator, add the human small cell lung cancer brain metastasis organoid culture medium after the matrix gel solidifies, add PBS to the remaining wells to reduce evaporation, and culture at 37°C, 5% CO2. Change the medium every 2-3 days. After 5-7 days of passaging, the cells can be used for downstream experiments.

[0053] Among them, the basal medium of the human small cell lung cancer brain metastasis organoid culture medium is advanced DMEM / F12 medium, supplemented with the following components: GlutaMax 2-5mM, HEPES 8-10mM, B271×-2×, SB202190500mM, Y-27632 5-10μM, A83-010.5μM, N-Acetyl-L-cysteine 0.5-2mM, Nicotinamide 5mM, recombinant human FGF725-100ng / ml, recombinant human FGF10100-500ng / ml, Human Noggin100-200ng / ml, Human R-Spondin-150-500ng / ml, Murine Wnt-3a 50-100ng / ml, FLI-062-10μM, penicillin-streptomycin 1×, Primocin 5~10μM.

[0054] In some embodiments, the tissue preservation solution in S1 is based on advanced DMEM / F12 culture medium, supplemented with the following reagents: penicillin 60 mg / L, streptomycin 0.1 g / L, Y-27632 10 μM, stored at 4° C., and used immediately upon preparation.

[0055] In some embodiments, the number of DPBS washings in S2 is 2 to 3 times.

[0056] In some embodiments, sterile scissors are used in S4 to mince the tissue sample into pieces of approximately 1 to 3 mm.

[0057] In some embodiments, the amount of 1×TryPLE Express added to S5 is 8 to 10 times the volume of the tissue fragments.

[0058] In some embodiments, the dissociation conditions in S5 are: the rotation speed is set to 60 rpm / min, and the dissociation time is 20 to 30 minutes.

[0059] In some embodiments, in S6, the conditions for centrifugation to remove the dissociation solution are 250×g and 5 min.

[0060] In some embodiments, in S7 , when sieving to remove undissociated tissue, the sieve is 100 μm.

[0061] In some embodiments, in S8, the centrifugation conditions for obtaining a single cell pellet are 250×g and 5 min.

[0062] In some embodiments, in S10, the inoculation method is to vertically inoculate 50 ul / well to the center of the bottom of the 24-well plate without touching the side walls of the 24-well plate.

[0063] In some embodiments, in S10, the method for adding the human small cell lung cancer brain metastasis organoid culture medium is to slowly add 700 μl / Well along the side wall.

[0064] In some embodiments, the method of passaging is as follows: remove the culture medium, add 1 ml of TrypLE Express to each well, gently pipette 5 to 10 times, transfer to a centrifuge tube rinsed with anti-adhesion solution, dissociate in a constant temperature shaker at 37°C for 15 minutes, centrifuge at 4°C, 250×g, 5 minutes, remove the supernatant, add DMEM / F12 for washing, centrifuge again and sieve, and perform subsequent operations as the primary culture, add matrix gel and culture on plates.

[0065] In some embodiments, the culturing method of the human small cell lung cancer brain metastasis organoids also includes freezing, and the freezing method is to select organoids with good growth conditions, remove the culture medium and add DPBS, gently blow with the tip of the gun to remove the mixture of organoids and matrix gel, transfer to a centrifuge tube and centrifuge, discard the supernatant and add pre-cooled freezing solution, blow and mix well, and then transfer to a freezing tube, 1 ml per tube, put it in a programmed cooling box, place it in a -80°C refrigerator, and transfer it to a liquid nitrogen tank for storage overnight.

[0066] In some embodiments, the culture method of the human small cell lung cancer brain metastasis organoid further includes resuscitation, and the resuscitation method is to preheat the human small cell lung cancer brain metastasis organoid culture medium and DMEM / F12 culture medium to 37°C in a water bath, take out the cryopreservation tube, quickly place it in a 37°C water bath to thaw it quickly, transfer it to 5-8 ml of DMEM / F12, and take 1 ml of DMEM / F12 again to clean the cryopreservation tube to reduce residue; after centrifugation, remove the supernatant to obtain the organoid precipitate.

[0067] The following describes it in detail with reference to specific preferred embodiments.

[0068] Example 1 Primary tissue culture

[0069] Obtain fresh surgical tissue samples, place them in 4°C pre-cooled tissue preservation solution, and transport them to the laboratory in an ice bath. Remove the tissue samples from the biosafety cabinet, add them to pre-cooled DPBS cleaning solution, and wash them three times. Place the tissue in a cell culture dish and trim the sample using sterile forceps and tissue scissors to remove necrotic tissue and capsule tissue. Transfer the tissue to a pre-cooled 1.5ml EP tube and use sterile scissors to mince the tissue sample into fragments of approximately 1-3mm. Transfer 1ml of tissue fragments to a 15ml centrifuge tube and add 10ml of 1×TryPLE After express, the cells were placed in a 37°C constant temperature shaker for dissociation, with the speed set at 60 rpm / min and the dissociation time set at 30 min. The dissociation solution was removed by centrifugation at 4°C, 250×g, and 5 min. The precipitate was washed with DMEM / F12 and passed through a 100μm sieve to remove undissociated tissue. The undissociated tissue was subjected to a secondary dissociation using the same method as before, with a dissociation time of 8 min. The single cell precipitate was obtained by centrifugation at 4°C, 250×g, and 5 min, and counted. According to the counting results, the advanced Resuspend the pellet to 2000-5000 cells per microliter in DMEM / F-12, calculate the amount of matrigel to be added according to the counting results, add matrigel (Corning#356231) at a ratio of 1:1, and inoculate into a 24-well plate preheated overnight. When inoculating, inoculate 50ul / Well vertically to the center of the bottom of the 24-well plate without touching the side walls of the 24-well plate. Transfer to a cell culture incubator and wait for 20 minutes. After the matrigel solidifies, add 700μl human small cell lung cancer brain metastasis organoid culture medium / Well. When adding human small cell lung cancer brain metastasis organoid culture medium, add it slowly along the side walls. Add PBS to the remaining wells to reduce evaporation. Incubate at 37°C and 5% CO2. The culture results are as follows: Figure 1 Group B and Figure 2 As shown in group B. After 5 to 7 days, the cells were subcultured and subsequently frozen or used for downstream experiments. The culture results of the P2 generation were shown in Figure 3 shown.

[0070] Among them, the subculture method is to remove the culture medium, add 1ml TrypLE Express to each well, gently pipette 5 to 10 times, transfer to a centrifuge tube rinsed with anti-adhesion solution, dissociate at 37°C constant temperature shaker for 15 minutes, centrifuge at 4°C, 250×g, 5 minutes, remove the supernatant, add DMEM / F12 for washing, centrifuge again and sieve, and the subsequent operation is the same as the primary culture, add matrix gel and then culture on the plate.

[0071] In this example, the tissue preservation solution is based on advanced DMEM / F12 culture medium, supplemented with the following reagents: penicillin 60 mg / L, streptomycin 0.1 g / L, Y-27632 10 μM, stored at 4° C., and used immediately after preparation.

[0072] In this example, the basal medium for the culture medium of human small cell lung cancer brain metastasis organoids is advanced DMEM / F-12, and the additives and final concentrations are as follows: GlutaMax 5mM; HEPES 10mM; B271×; SB202190500nM; Y-2763210μM; A83-010.5μM; N-Acetyl-L-cysteine 1.25mM; Nicotinamide 5mM; recombinant human FGF 725ng / ml; recombinant human FGF10 100ng / ml; human Noggin 200ng / ml; human R-Spondin-1500ng / ml; Murine Wnt-3a 100ng / ml; FLI-06 10μM; Primocin 10μM; and penicillin-streptomycin 0.1g / L.

[0073] Example 2 Cryopreservation

[0074] The freezing method is to select organoids with good growth conditions, remove the culture medium and add DPBS, gently blow the tip of the gun to remove the mixture of organoids and matrix gel, transfer to a centrifuge tube and centrifuge, discard the supernatant and add pre-cooled freezing solution ( CS10), pipet and mix thoroughly, transfer to cryopreservation tubes, 1 ml per tube, place in a programmed cooling box, place in a -80°C refrigerator, and transfer to a liquid nitrogen tank for storage overnight.

[0075] Example 3 Resuscitation

[0076] The thawing method is to preheat CGM and DMEM / F12 culture medium to 37℃ in a water bath, remove the cryovial, quickly place it in a 37℃ water bath to thaw quickly, transfer it to 5-8ml of DMEM / F12, and take 1ml of DMEM / F12 to clean the cryovial to reduce residue; after centrifugation, remove the supernatant to obtain the organoid pellet, and the subsequent operation is the same as before. Among them, the culture results after P1 generation cryopreservation thawing are as follows Figure 4 shown.

[0077] Comparative Example

[0078] The culture method is derived from the patent "A culture medium and modeling method for difficult-to-culture lung cancer brain metastasis organoids" (CN202311835378.9). The culture medium is prepared as follows: Advanced DMEM / F12 50%, NeurobasalTM 50%, Glutama 1%, Primocin 200μg / ml, penicillin-streptomycin 200U / ml and 200mg / ml respectively, R-Spondin 500ng / ml, Noggin 500ng / ml, EGF 100ng / ml, FGF10200ng / ml, FGF750ng / ml, insulin 5μg / ml, B272%, N-AcetylCysteine 2mM, Nicotinamide 20mM, A83-011μM, Y2763220μM. The tissue digestion enzyme was prepared as follows: DMEM basal medium, 20% fetal bovine serum, 2 mg / ml type IV collagenase, Y2763220 μM, primocin 100 μg / ml, 100 U / ml penicillin / 100 mg / ml streptomycin.

[0079] Obtain fresh surgical tissue samples, place them in 4-degree pre-cooled tissue preservation solution, and transport them to the laboratory in an ice bath; remove the tissue samples from the biosafety cabinet, add them to pre-cooled DPBS cleaning solution, and wash them three times; place the tissue in a cell culture dish, use sterile tweezers and tissue scissors to trim the sample tissue, and remove necrotic tissue and capsule tissue; transfer the tissue to a pre-cooled 1.5ml EP tube, and use sterile scissors to cut the tissue sample into fragments of approximately 1 to 3mm; transfer 1ml of tissue fragments to a 50ml centrifuge tube, add 10ml of prepared tissue digestion enzyme, and place it in a 37°C, 5% CO2 incubator for digestion for 3 hours, then add 20ml Terminate digestion with Advanced DMEM / F12 at 4°C, pipette repeatedly to disperse cells, centrifuge at 300×g for 5 minutes, discard supernatant to obtain cell pellet, add 20% matrigel according to the pellet volume, mix thoroughly, and add to preheated 24-well plate. After solidification in the incubator for 20 minutes, add 500 μl of organoid culture medium, and change the medium every 3 days. Figure 1 Group A and Figure 2 Shown in Group A.

[0080] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. All modifications, equivalent substitutions, improvements, etc. within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A culture medium for human small cell lung cancer brain metastasis organoids, characterized in that The culture medium is based on advanced DMEM / F12 medium and is supplemented with the following components: GlutaMax 2-5 mM, HEPES 8-10 mM, B27 1×-2×, SB202190 500 mM, Y-27632 5-10 μM, A83-0 10.5 μM, N-Acetyl-L-cysteine 0.5-2 mM, Nicotinamide 5 mM, recombinant human FGF 7 2 5-100 ng / ml, recombinant human FGF 10 100-500 ng / ml, Human Noggin 100-200 ng / ml, Human R-Spondin-1 50-500 ng / ml, Murine Wnt-3a 50-100 ng / ml, FLI-06 2-10 μM, penicillin-streptomycin 1×, and Primocin 5-10 μM.

2. A method for culturing human small cell lung cancer brain metastasis organoids, characterized in that: These include: S1. Place the fresh tissue sample in 4°C pre-cooled tissue preservation solution and transport it to the laboratory on ice. S2. Remove the tissue sample from the biosafety cabinet and wash it in pre-chilled DPBS. S3. Place the tissue in a cell culture dish and trim the sample using sterile forceps and tissue scissors to remove necrotic tissue and encapsulation. S4. Transfer the tissue to a pre-chilled EP tube and mince the tissue sample using sterile scissors. S5. Transfer the tissue fragments to a centrifuge tube, add 1× TryPLE Express Dissociation Buffer, and place in a 37°C incubator for dissociation. S6. Centrifuge at 4°C to remove dissociation buffer; S7. Wash the pellet with DMEM / F12 and sieve to remove undissociated tissue. S8. Centrifuge at 4°C to obtain single cell pellets and count them. S9. Resuspend the pellet to 2000–5000 cells per μL in advanced DMEM / F-12 according to the cell count result. Resuspend the cell suspension in Matrigel at a 1:1 volume ratio and place on ice. S10. Remove the preheated 24-well plate, inoculate the cell resuspension into the 24-well plate, transfer the plate to a cell culture incubator, and add the human small cell lung cancer brain metastasis organoid culture medium described in claim 1 after the matrix gel solidifies. Add PBS to the remaining wells to reduce evaporation. Culture at 37°C and 5% CO2, changing the medium every 2 to 3 days. After 5 to 7 days of passaging, the cells can be used for downstream experiments.

3. The method according to claim 2, characterized in that The tissue preservation solution in S1 is based on advanced DMEM / F12 culture medium and supplemented with the following reagents: penicillin 60 mg / L, streptomycin 0.1 g / L, Y-27632 10 μM. It is stored at 4°C and used immediately after preparation.

4. The method according to claim 2, characterized in that The number of DPBS washes in S2 was 2 to 3 times.

5. The method according to claim 2, characterized in that In S4, the tissue sample is minced into pieces of approximately 1 to 3 mm using sterile scissors.

6. The method according to claim 2, characterized in that The amount of 1×TryPLE Express added to S5 was 8 to 10 times the volume of the tissue fragments, and the dissociation conditions were: the rotation speed was set at 60 rpm / min, and the dissociation time was 20 to 30 min.

7. The method according to claim 2, characterized in that In S6, the centrifugation conditions for removing the dissociation solution were 250×g for 5 minutes; in S7, when sieving to remove undissociated tissue, the cells were sieved through a 100 μm sieve; in S8, the centrifugation conditions for obtaining a single cell pellet were 250×g for 5 minutes.

8. The method according to claim 2, characterized in that In S10, the inoculation method is to vertically inoculate 50ul / Well into the center of the bottom of a 24-well plate without touching the side walls of the 24-well plate; the method for adding the human small cell lung cancer brain metastasis organoid culture medium is to slowly add 700μl / Well along the side walls; the method for subculture treatment is as follows: remove the culture medium, add 1ml of TrypLE Express to each well, gently pipette 5-10 times, transfer to a centrifuge tube rinsed with anti-adhesion solution, dissociate on a constant temperature shaker at 37°C for 15 minutes, centrifuge at 4°C, 250×g, for 5 minutes, remove the supernatant, add DMEM / F12 for washing, centrifuge again, and sieve. Subsequent operations are the same as primary culture, and after adding matrix gel, plate culture is performed.

9. The method according to claim 2, characterized in that The culture method of the human small cell lung cancer brain metastasis organoids also includes freezing. The freezing method comprises selecting organoids with good growth conditions, removing the culture medium and adding DPBS, gently blowing with the tip of a gun to remove the mixture of organoids and matrix gel, transferring the mixture to a centrifuge tube and centrifuging, discarding the supernatant, adding pre-cooled freezing solution, blowing and mixing, and then transferring the mixture to a freezing tube, 1 ml per tube, placing the tube in a programmed cooling box, placing it in a -80°C refrigerator, and transferring it to a liquid nitrogen tank for storage overnight.

10. The method according to claim 2, characterized in that The culture method of the human small cell lung cancer brain metastasis organoids also includes resuscitation, and the resuscitation method is to preheat the human small cell lung cancer brain metastasis organoid culture medium and DMEM / F12 culture medium to 37°C in a water bath, remove the cryopreservation tube, quickly place it in a 37°C water bath to quickly thaw it, transfer it to 5-8 ml of DMEM / F12, and take 1 ml of DMEM / F12 again to clean the cryopreservation tube to reduce residue; after centrifugation, remove the supernatant to obtain the organoid precipitate.

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