Preparation method and kit for organoid culture medium

Abstract

The invention relates to the technical field of cell culture, in particular to a preparation method and kit for an organoid culture medium, and the preparation method for the organoid culture medium comprises the following steps: sequentially mixing serum-free B27, nicotinamide and other components to obtain a first solution; adding Wnt3a, RSPO1 and Noggin with corresponding concentrations on the basis of whether the organoid has KRAS mutation or not to obtain a second solution; selectively adding an N-2 additive or estradiol based on the type of the tumor organoid to obtain a third solution; and finally, DMEM (Dulbecco Modified Eagle Medium) based and Advanced DMEM / F12 are added to obtain the organoid culture medium. According to the invention, the concentration and combination of key components in the culture medium can be dynamically adjusted according to three key characteristics of the type of the tumor organoid, whether the tumor organoid is a KRAS mutant type or not and the content of tumor parenchymal cells. The kit comprises an organoid culture medium, a rinsing solution, a tissue digestion solution, a neutralization solution and a passage digestion solution. The defect that an existing fixed formula culture medium is difficult to adapt to sample heterogeneity is overcome, and the success rate and culture stability of organoid construction are improved.

Description

Technical Field

[0001] This invention relates to the field of cell culture technology, and in particular to a method and kit for preparing organoid culture medium. Background Technology

[0002] Organoids, as three-dimensional in vitro models simulating the structure and function of organs in vivo, possess core value in overcoming the limitations of traditional two-dimensional cell culture and achieving high-fidelity simulation of the physiological and pathological characteristics of human tissues. Since its breakthrough at the beginning of this century, tumor organoid culture technology has undergone several stages of development, from proof-of-concept to model expansion, and now to standardization and clinical translation. This technology has been applied in disease modeling, drug screening, precision medicine, and regenerative therapy. Improving the success rate of organoid construction, controlling passage stability, and refining imaging evaluation are the core technological pillars driving its transition from laboratory to clinical application.

[0003] However, despite progress in automation and phenotypic analysis, one of the core bottlenecks in current tumor organoid culture technology—the universality and static nature of culture medium formulations—remains fundamentally unresolved. Existing commercially available or literature-reported culture medium regimens mostly employ fixed-component designs, lacking a precise response to the inherent heterogeneity of different tumor samples. Specifically, existing technologies mainly suffer from the following shortcomings: 1. Insufficient tissue specificity: Different cancer types rely on differentiated growth signals, but universal formulas cannot be precisely adapted, resulting in low culture efficiency for some types.

[0004] 2. Genotype-specific loss: For samples carrying specific driver mutations, their signaling pathway dependence is altered, and the concentration of fixation factors cannot provide optimal support, affecting culture stability.

[0005] 3. Inadequate support for low-purity samples: For trace samples such as those obtained by puncture with low tumor parenchymal cell content, the signal and nutritional support provided by the general culture medium is insufficient, leading to clone formation failure.

[0006] Therefore, developing a culture medium preparation method and kit that can be dynamically optimized based on three key dimensions—tumor type, gene mutation status, and sample cell content—has become an urgent need to promote the standardization and high-success-rate clinical translation of this technology. Summary of the Invention

[0007] Therefore, the present invention provides a method and kit for preparing organoid culture medium to overcome the problems of low organoid construction success rate caused by insufficient tissue specificity, lack of genotype specificity and poor support for low-purity samples in the prior art.

[0008] To achieve the above objectives, the present invention provides a method for preparing an organoid culture medium, comprising the following steps: Step S1: Mix 50X serum-free B27, 10mM nicotinamide, 1.25mM N-acetylcysteine, Primocin, 10μM Y-27632 2HCl, 0.05ng / mL Animal-Free KGF-2 / FGF-10, 10nM gastrin, 10μM MSB202190, 10μM trichosine, 3μM dexamethasone and 1μM A8301 to obtain a first solution; Step S2: Add Wnt3a, RSPO1 and Noggin to the first solution, and based on the result that the tumor organoids are of the KRAS mutation type, increase the concentration of Wnt3a, RSPO1 and Noggin to obtain a second solution; Step S3: Based on the type of tumor organoid, determine whether to add N-2 additive or estradiol to the second solution to obtain a third solution; Step S4: Add DMEM basic and Advanced DMEM / F12 to the third solution to obtain organoid culture medium.

[0009] Further, in step S1, the amount of 50X serum-free B27 added is 1 mL, the amount of 10 mM nicotinamide added is 500 μL, the amount of 1.25 mM N-acetylcysteine ​​added is 125 μL, the amount of Primocin added is 100 μL, the amount of 10 μM Y-27632 2HCl added is 50 μL, the amount of 0.05 ng / mL Animal-Free KGF-2 / FGF-10 added is 5 μL, the amount of 10 nM gastrin added is 5 μL, the amount of 10 μM SB202190 added is 5 μL, the amount of 10 μM trichosin added is 50 μL, the amount of 3 μM dexamethasone added is 50 μL, and the amount of 1 μM MA8301 added is 2 μL.

[0010] Further, in step S1, the concentration of Primocin in the first solution is 100 μg / mL.

[0011] Further, in step S2, the concentrations of Wnt3a, RSPO1, and Noggin are all 0.05 μg / mL, and the addition amounts of Wnt3a, RSPO1, and Noggin are all 25 μL.

[0012] Furthermore, step S2 also includes increasing the concentrations of Wnt3a, RSPO1, and Noggin to 0.1 μg / mL based on the result that the tumor organoid is a KRAS mutant.

[0013] Furthermore, step S3 also includes, based on the result that the tumor organoid is a lung cancer organoid, determining to add 500 μL of N-2 additive to the second solution.

[0014] Furthermore, step S3 also includes, based on the result that the tumor organoid is a breast cancer organoid, determining to add 5 μL of 5 μg / mL estradiol to the second solution.

[0015] Furthermore, step S4 also includes adding 30 mL of DMEM basic and 18 mL of Advanced DMEM / F12.

[0016] Furthermore, step S4 also includes adjusting the amount of DMEM basic added to 20 mL and the amount of Advanced DMEM / F12 added to 13 mL based on the result that the content of tumor parenchymal cells is lower than the preset content, and adding 15 mL of fibroblast supernatant to the third solution.

[0017] Another aspect of the present invention provides a kit for culturing organoids, comprising: the organoid culture medium, a rinsing solution, a tissue digestion solution, a neutralizing solution, and a passage digestion solution, wherein the rinsing solution contains bovine serum albumin and phosphate buffer, the tissue digestion solution contains a collagenase combination, the neutralizing solution contains a basal culture medium, a buffer, supplemented amino acids, and antibiotics, and the passage digestion solution contains a protease and a metal ion chelating agent.

[0018] Compared with existing technologies, the beneficial effects of this invention are as follows: The organoid culture medium preparation method and kit provided by this invention establish a method for dynamically optimizing culture medium components based on three key characteristics: tumor organoid type, gene mutation status, and parenchymal cell content. By introducing conditional adjustments in the preparation steps, it achieves precise matching of culture medium components and concentrations to specific culture requirements, thereby overcoming the shortcomings of traditional fixed-formulation culture media that are unable to cope with the heterogeneity of clinical samples, resulting in low construction success rates. Furthermore, by pre-packaging the organoid culture medium and related reagents into individual reagent bottles in a commercialized kit format, the operation process is simplified, time and cost consumption are reduced, and the standardization and reproducibility of experiments are improved. This provides reliable and efficient technical support for the application of tumor organoid technology in disease modeling, drug screening, and clinical translation.

[0019] Furthermore, by precisely controlling the amount of each basic component added in step S1, a nutritionally balanced and signal-stable basic culture system was constructed, achieving multiple synergistic supports for cell survival, proliferation, anti-oxidation, anti-apoptosis, and phenotype maintenance. This provides a reliable starting basis for subsequent dynamic optimization targeting tumor type, gene mutation, and cell content.

[0020] Furthermore, by limiting the concentration of Primocin to 100 μg / mL, bacterial and fungal contamination was effectively inhibited while toxic damage to primary tumor cells was avoided, achieving a balance between the cleanliness of the culture system and cell safety, thereby reducing the culture failure rate caused by microbial contamination.

[0021] Furthermore, by setting the baseline concentrations of Wnt3a, RSPO1, and Noggin to 0.05 μg / mL, conventional Wnt signal activation intensity and BMP signal inhibition levels applicable to most tumor organoids were provided, achieving basic maintenance of tumor stem cell characteristics and thus establishing a standardized starting point for subsequent concentration adjustments targeting specific mutation types.

[0022] Furthermore, by doubling the concentrations of Wnt3a, RSPO1, and Noggin in KRAS-mutant tumors, the activation level of the Wnt / β-catenin pathway was enhanced and the inhibition of BMP signaling was strengthened, satisfying the abnormal dependence of KRAS-mutant cells on continuous self-renewal signals, thereby improving the success rate of mutant organoid construction and growth viability.

[0023] Furthermore, by adding N-2 additive to lung cancer organoids, specific components such as transferrin, insulin, and sodium selenite required for neuronal development and survival were supplemented, thereby significantly improving the proliferation efficiency and culture stability of lung cancer organoids.

[0024] Furthermore, by adding estradiol to breast cancer organoids, a key activating ligand for the estrogen receptor signaling pathway was provided, mimicking the hormone-sensitive microenvironment in vivo and satisfying the dependence of estrogen receptor-positive breast cancer cells on hormone signals, thereby effectively promoting the survival and expansion of this type of breast cancer organoid.

[0025] Furthermore, by setting the regular addition amounts of DMEM basic and Advanced DMEM / F12 to 30 mL and 18 mL respectively, a balanced basic nutrient supply system was constructed. DMEM basic provides abundant amino acids and vitamins, while Advanced DMEM / F12 supplements more nutrients and buffering capacity, thereby meeting the basic metabolic needs of most tumor organoids.

[0026] Furthermore, by adjusting the basal culture medium ratio and adding fibroblast supernatant rich in various growth factors and cytokines to low-purity samples with less than 20% tumor parenchymal cells, the paracrine support effect in the tumor microenvironment was simulated, providing additional survival and proliferation signals for a small number of tumor cells, thereby significantly improving the organoid clonal formation efficiency of low-purity samples. Attached Figure Description

[0027] Figure 1 This is a flowchart illustrating the steps of a method for preparing an organoid culture medium according to an embodiment of the present invention; Figure 2 Bright-field micrographs of cultured organoids in Example 1 and Comparative Examples 1-4 of this invention; Figure 3 Bright-field micrographs of cultured organoids in Example 2 and Comparative Example 5 of this invention; Figure 4 Bright-field micrographs of cultured organoids in Example 3 and Comparative Example 6 of this invention; Figure 5 Bright-field micrographs of cultured organoids in Example 4 and Comparative Example 7 of this invention; Figure 6 Bright-field micrographs of cultured organoids in Example 5 and Comparative Examples 8-9 of this invention; Figure 7 Bright-field micrographs of cultured organoids in Example 6 and Comparative Example 10 of this invention; Figure 8 Bright-field micrographs of cultured organoids in Example 7 and Comparative Example 11 of this invention; Figure 9 Bright-field micrographs of cultured organoids in Example 8 and Comparative Example 12 of this invention; Figure 10 Bright-field micrographs of cultured organoids in Example 9 and Comparative Examples 13-14 of this invention; Figure 11 Bright-field micrographs of cultured organoids in Example 10 and Comparative Example 15 of this invention. Detailed Implementation

[0028] To make the objectives and advantages of the present invention clearer, the present invention will be further described below with reference to embodiments; it should be understood that the specific embodiments described herein are merely for explaining the present invention and are not intended to limit the present invention.

[0029] Preferred embodiments of the present invention will now be described with reference to the accompanying drawings. Those skilled in the art should understand that these embodiments are merely illustrative of the technical principles of the present invention and are not intended to limit the scope of protection of the present invention.

[0030] Please see Figure 1As shown, Figure 1 This is a flowchart illustrating the steps of a method for preparing an organoid culture medium according to an embodiment of the present invention.

[0031] The method for preparing organoid culture medium according to an embodiment of the present invention, as shown in Table 1 (organoid culture medium formulation), includes the following steps: Step S1: Mix 50X serum-free B27, 10mM nicotinamide, 1.25mM N-acetylcysteine, Primocin, 10μM Y-27632 2HCl, 0.05ng / mL Animal-Free KGF-2 / FGF-10, 10nM gastrin, 10μM MSB202190, 10μM trichosine, 3μM dexamethasone and 1μM A8301 to obtain the first solution; Step S2: Add Wnt3a, RSPO1 and Noggin to the first solution, and increase the concentration of Wnt3a, RSPO1 and Noggin based on the result that the tumor organoids are of the KRAS mutation type to obtain the second solution; Step S3: Based on the type of tumor organoid, determine whether to add N-2 additive or estradiol to the second solution to obtain the third solution; Step S4: Add DMEM basic and Advanced DMEM / F12 to the third solution to obtain organoid culture medium.

[0032] Specifically, in step S1, the following amounts were added: 1 mL of 50X serum-free B27, 500 μL of 10 mM nicotinamide, 125 μL of 1.25 mM N-acetylcysteine, 100 μL of Primocin, 50 μL of 10 μM Y-27632 2HCl, 5 μL of 0.05 ng / mL Animal-Free KGF-2 / FGF-10, 5 μL of 10 nM gastrin, 5 μL of 10 μM SB202190, 50 μL of 10 μM trichosin, 50 μL of 3 μM dexamethasone, and 2 μL of 1 μM A8301.

[0033] Understandably, the components added in step S1 collectively constitute the basic nutritional and signal support system for organoid culture. Among them, B27, as a core nutritional supplement, provides abundant antioxidants, vitamins, and hormones, laying the foundation for cell survival and proliferation; nicotinamide participates in energy metabolism and DNA repair, maintaining cell viability; N-acetylcysteine ​​scavenge reactive oxygen species, reducing oxidative damage; Primocin ensures the sterility of the culture system, avoiding culture failure caused by contamination; Y-27632 inhibits apoptosis, improving the survival rate of primary and passaged cells; KGF-2 / FGF-10 promotes epithelial cell proliferation, maintaining stemness; gastrin stimulates the growth of digestive tract-derived cells; SB202190 inhibits the p38 pathway, preventing cell differentiation; trichosine increases cAMP levels, regulating cell function; dexamethasone stabilizes the cell membrane, inhibits inflammatory responses, and promotes differentiation balance; A8301 blocks TGF-β signaling, inhibiting mesenchymal transition and maintaining epithelial characteristics. These components work synergistically to provide a stable environment for subsequent dynamic adjustments to specific tumor types, gene mutations, and cell content, ensuring the successful construction and long-term culture of organoids.

[0034] Specifically, in step S1, the concentration of Primocin in the first solution is 100 μg / mL.

[0035] Understandably, this concentration of Primocin can effectively inhibit bacterial and fungal contamination while avoiding the toxicity of excessively high concentrations of antibiotics to primary tumor cells, thus providing a clean and safe growth environment for the initial culture of organoids, and is especially suitable for the risk of microbial contamination that may exist in clinically derived samples.

[0036] Specifically, step S2 also includes increasing the concentrations of Wnt3a, RSPO1, and Noggin to 0.1 μg / mL based on the result that the tumor organoids are KRAS mutants.

[0037] Understandably, KRAS mutant tumor cells typically exhibit enhanced dependence on the Wnt signaling pathway and require stronger BMP signaling inhibition to maintain their stemness and proliferative capacity. Increasing the concentrations of Wnt3a, RSPO1, and Noggin from 0.05 μg / mL to 0.1 μg / mL more effectively activated the Wnt / β-catenin pathway and inhibited BMP signaling, thereby meeting the abnormal demand of KRAS mutant cells for continuous self-renewal signals and significantly improving the success rate and growth viability of this type of mutant organoid.

[0038] Specifically, step S3 also includes determining to add 500 μL of N-2 additive to the second solution based on the result that the tumor organoid is a lung cancer organoid.

[0039] Understandably, N-2 additive contains ingredients such as transferrin, insulin, and sodium selenite, which can provide specific nutritional and signal support required for neuronal development and survival. In addition, adding N-2 can mimic the in vivo microenvironment, promote the balance of proliferation and differentiation of lung cancer organoids, and thus improve the culture effect.

[0040] Specifically, step S3 also includes, based on the result that the tumor organoid is a breast cancer organoid, determining to add 5 μL of 5 μg / mL estradiol to the second solution.

[0041] It is understandable that estradiol is a key activating ligand in the estrogen receptor signaling pathway. For estrogen receptor-positive breast cancer, the addition of estradiol can mimic the in vivo hormonal environment, activate downstream proliferative signals, and thus effectively support the survival and expansion of this type of breast cancer organoids, improving the success rate of culture.

[0042] Specifically, step S4 also includes adding 30 mL of DMEM basic and 18 mL of Advanced DMEM / F12.

[0043] Understandably, this basal culture medium ratio provides balanced nutritional support for most tumor organoids. DMEM basic is rich in amino acids and vitamins, while Advanced DMEM / F12 contains even more nutrients and a buffer system. The specific ratio of the two can meet the metabolic needs of different types of tumor cells and provide a stable basal environment for organoid growth.

[0044] Specifically, step S4 also includes adjusting the amount of DMEM basic added to 20 mL and the amount of Advanced DMEM / F12 added to 13 mL based on the result that the content of tumor parenchymal cells is lower than the preset content, and adding 15 mL of fibroblast supernatant to the third solution.

[0045] Understandably, when the tumor parenchymal cell content is below 20%, the number of starting cells is scarce, requiring stronger growth support. By adjusting the basal culture medium volume and adding fibroblast supernatant rich in various growth factors and cytokines, the paracrine effect in the tumor microenvironment can be simulated, providing additional survival and proliferation signals for a small number of tumor cells, thereby improving the organoid clone formation efficiency of low-purity samples. This enhancement strategy can significantly improve the culture success rate and avoid culture failure due to insufficient starting cells.

[0046] Table 1 Organoid Culture Medium Formulation Table ; The kit for culturing organoids according to embodiments of the present invention includes: The first reagent bottle contains organoid culture medium; The second reagent bottle contains a washing solution, which includes bovine serum albumin and phosphate buffer. The third reagent bottle contains tissue digestion solution, which includes a collagenase complex. The fourth reagent bottle contains a neutralization solution, which includes basal culture medium, buffer, supplemented amino acids, and antibiotics. The fifth reagent bottle contains a subculture digest, which includes protease and metal ion chelating agents.

[0047] Specifically, 0.1g of bovine serum albumin powder was weighed and added to 100mL of PBS buffer to obtain the washing solution.

[0048] Specifically, collagenase XI, collagenase IV, and collagenase II are mixed in a 2:1:1 ratio to obtain a tissue digestion solution.

[0049] Specifically, DMEM-F12, 1M HEPES, GlutaMAX, and Antibiotic-Antimycotic are mixed to obtain a neutralized solution.

[0050] Specifically, recombinant trypsin-like protease and 1 mM EDTA were mixed to obtain a passaged digest.

[0051] Example 1

[0052] Sample source: Surgically removed pancreatic cancer tissue.

[0053] Preparation process: (1) Culture medium preparation: Pancreatic cancer organoid culture medium was prepared according to the organoid culture medium formula table shown in Table 1.

[0054] (2) Organoid construction: a. Preliminary cleaning and mincing: Place the tissue sample in a disposable 6cm petri dish, rinse the tissue surface with the rinsing solution from the second reagent bottle, and mince the tissue sample into 1mm pieces using sterile scissors. 3 Obtain shredded samples from blocks of approximately the same size (left and right). b. Digestion: Transfer the minced tissue into a 15mL centrifuge tube, add the tissue digestion solution from the third reagent bottle, and digest at 150rpm for 20-25 minutes in a shaker at 37℃. When the tissue sample in the centrifuge tube decreases and the liquid becomes turbid, add an equal volume of neutralizing solution from the fourth reagent bottle to stop the digestion and obtain the digestion solution. c. Sieving and grinding: Pour the above digestion solution into a 100μm filter screen, and while grinding in a circular motion using the piston of a brand new sterile 10mL syringe, add the neutralization solution from the fourth reagent bottle. Collect the cells that have passed through the filter screen into a centrifuge tube to obtain a cell suspension. d. Centrifugation and erythrocyte lysis: Centrifuge the above cell suspension at 1400 rpm for 5 min, discard the supernatant, add 2 mL of erythrocyte lysis buffer to the precipitate, mix well by pipetting and aspiration, let stand for 5 min, centrifuge at 1400 rpm for 5 min, discard the supernatant, obtain tumor parenchymal cells, and detect their content. e. Seeding gel: Mix the above-mentioned tumor parenchymal cells with pre-cooled liquid Matrigel, mix thoroughly by blowing and aspiration, and then seed them into the wells of a 24-well plate. Incubate in a 5% CO2 incubator at 37°C for 25 minutes until solidification. f. Culture: Take out the 24-well plate, add 600 μL of the corresponding organoid culture medium, and continue to culture in a 5% CO2 incubator at 37°C.

[0055] (3) Organoid passage: a. Washing: After 48 hours of culture, remove the 24-well plate, discard the old organoid culture medium, and gently rinse twice with the washing solution from the second reagent bottle; b. Digestion: Add 1 mL of the passage digestion solution from the fifth reagent bottle to each well of the 24-well plate, and repeatedly pipette until the gel is melted and uniform to obtain a resuspension. Transfer the resuspension to a 15 mL centrifuge tube and incubate at 37°C for 10-15 min. Then add 1 mL of the neutralization solution from the fourth reagent bottle, centrifuge at 400G for 5 min, discard the supernatant, and obtain the cell pellet. c. Seeding gel: Mix the above cell pellet with pre-cooled liquid Matrigel, gently blow and aspirate to mix, then seed into the wells of a new 24-well plate and incubate at 37°C for 25 minutes in a 5% CO2 incubator until solidified. d. Culture: Take out the new 24-well plate, add 600 μL of the corresponding new organoid culture medium, and continue to culture in a 5% CO2 incubator at 37°C. Change the medium and passage the plate every 48 hours.

[0056] Comparative Example 1 Sample source: Same as Example 1.

[0057] Preparation process: (1) Culture medium preparation: A universal culture medium was prepared, which differed from the pancreatic cancer organoid culture medium in Example 1 in that it did not contain the following components: B27, nicotinamide, fenestrated linolenic acid, and dexamethasone. Other components and their amounts were the same as in Example 1.

[0058] (2) Organoid construction: Same as in Example 1.

[0059] (3) Organoid passage: Same as in Example 1.

[0060] Comparative Example 2 Sample source: Same as Example 1.

[0061] Preparation process: (1) Culture medium preparation: Unlike the pancreatic cancer organoid culture medium in Example 1, 500 μL of N-2 additive was added. Other components and amounts were the same as in Example 1.

[0062] (2) Organoid construction: Same as in Example 1.

[0063] (3) Organoid passage: Same as in Example 1.

[0064] Comparative Example 3 Sample source: Same as Example 1.

[0065] Preparation process: (1) Culture medium preparation: Unlike the pancreatic cancer organoid culture medium in Example 1, 5 μL of estradiol was added. Other components and amounts were the same as in Example 1.

[0066] (2) Organoid construction: Same as in Example 1.

[0067] (3) Organoid passage: Same as in Example 1.

[0068] Comparative Example 4 Sample source: Same as Example 1.

[0069] Preparation process: (1) Culture medium preparation: Unlike the pancreatic cancer organoid culture medium in Example 1, 500 μL of N-2 additive and 5 μL of estradiol were added. Other components and amounts were the same as in Example 1.

[0070] (2) Organoid construction: Same as in Example 1.

[0071] (3) Organoid passage: Same as in Example 1.

[0072] Table 2. Cell counts in Example 1 and Comparative Examples 1-4 ; Results and Analysis: As shown in Table 2 and Figure 2 As shown in Table 2, the cell counts for Example 1 and Comparative Examples 1-4 are as follows. Figure 2Bright-field micrographs of the cultured organoids from Example 1 and Comparative Examples 1-4 show that different culture medium components significantly affect the culture efficacy of pancreatic cancer organoids. The pancreatic cancer organoid culture medium used in Example 1 exhibited the best culture effect, yielding 3.37 × 10⁻⁶ cells. 5 / mL. From Figure 2 As can be seen from the bright-field micrograph, the organoids cultured in Example 1 exhibit a typical three-dimensional structure with regular and full morphology, clear edges, and a large number of organoids of uniform size.

[0073] In contrast, Comparative Example 1 (universal culture medium) performed extremely poorly, with a cell count of only 8.36 × 10⁻⁶. 3 The cell count in Comparative Example 1 was significantly lower than that in Example 1. This culture medium lacked B27, nicotinamide, estradiol, and dexamethasone. B27 is an important nutritional supplement, rich in antioxidants and vitamins, which promotes the survival and growth of nerve cells and various stem cells; nicotinamide is a derivative of vitamin B3, involved in cellular energy metabolism and DNA repair; estradiol can increase intracellular cAMP levels, affecting cell signaling; dexamethasone is a glucocorticoid that helps maintain cell culture homeostasis, inhibits apoptosis, and promotes cell differentiation. The absence of these key components is likely the main reason for the extremely low culture efficiency in Comparative Example 1, confirming their necessity for the initial culture of pancreatic cancer organoids. Although Comparative Examples 2, 3, and 4 added N-2 additive (Comparative Example 2), estradiol (Comparative Example 3), or both (Comparative Example 4) respectively to the culture medium based on Example 1, the culture results were all inferior to Example 1, with cell numbers of 9.52 × 10⁻⁶ cells. 4 / mL, 8.33×10 4 / mL and 7.62×10 4 / mL. From Figure 2 As can be seen, although organoid formation was observed in Comparative Examples 2-4, the number was significantly less than in Example 1, and some organoids exhibited irregular morphology, loose structure, or blurred edges. This suggests that the additional addition of N-2 or estradiol may have disrupted the optimized culture medium ratio, interfering with the normal development of organoids. In summary, the culture medium formulation of Example 1 has significant advantages in promoting the growth of pancreatic cancer organoids and maintaining good morphology.

[0074] Example 2

[0075] Sample source: Surgically removed pancreatic cancer tissue, which was confirmed by sequencing to be KRAS mutant.

[0076] Preparation process: (1) Culture medium preparation: Based on the pancreatic cancer organoid culture medium in Example 1, the concentrations of Wnt3a, RSPO1 and Noggin were adjusted to 0.1 μg / mL to prepare pancreatic cancer KRAS mutant organoid culture medium.

[0077] (2) Organoid construction: Same as in Example 1.

[0078] (3) Organoid passage: Same as in Example 1.

[0079] Comparative Example 5 Sample source: Same as Example 2.

[0080] Preparation process: (1) Culture medium preparation: Same as in Example 1, except that the concentrations of Wnt3a, RSPO1 and Noggin were not adjusted compared to the pancreatic cancer KRAS mutant organoid culture medium in Example 2.

[0081] (2) Organoid construction: Same as in Example 1.

[0082] (3) Organoid passage: Same as in Example 1.

[0083] Table 3 Cell counts in Example 2 and Comparative Example 5 ; Results and Analysis: As shown in Table 3 and Figure 3 As shown in Table 3, the cell counts for Example 2 and Comparative Example 5 are as follows. Figure 3 Bright-field micrographs of the cultured organoids from Example 2 and Comparative Example 5 are shown. For KRAS-mutant pancreatic cancer samples, the organoid culture results of Example 2 were far superior to those of Comparative Example 5, with a nearly thousand-fold difference in cell count. Figure 3 Bright-field micrographs show that in Example 2, organoids formed densely, exhibiting typical pancreatic cancer organoid morphology with clear, three-dimensional structures and good growth. In contrast, in Comparative Example 5, almost no obvious organoid formation was observed, with only a few cell fragments or unformed cell clusters, indicating a near-complete culture failure. This demonstrates that precisely adjusting the concentrations of Wnt3a, RSPO1, and Noggin in the culture medium is crucial for KRAS mutations. KRAS-mutant tumor organoids have specific microenvironment signaling requirements: optimized Wnt signaling helps maintain their stemness and proliferative capacity, while sufficient Noggin inhibition of BMP signaling prevents cell differentiation. Comparative Example 5, by not adjusting the concentrations of these three key factors, failed to meet the specific needs of the mutant cells, resulting in the inability to support normal organoid formation. In conclusion, optimizing the culture medium composition for KRAS-mutant organoids can significantly improve the success rate of constructing this type of pancreatic cancer organoid.

[0084] Example 3

[0085] Sample source: Surgically removed pancreatic cancer tissue with a tumor parenchymal cell content of less than 20%.

[0086] Preparation process: (1) Culture medium preparation: Based on the pancreatic cancer organoid culture medium in Example 1, the addition amounts of DMEM basic and Advanced DMEM / F12 were adjusted to 20 mL and 13 mL respectively, and 15 mL of fibroblast supernatant was added to prepare pancreatic cancer organoid enhanced culture medium.

[0087] (2) Organoid construction: Same as in Example 1.

[0088] (3) Organoid passage: Same as in Example 1.

[0089] Comparative Example 6 Sample source: Same as Example 3.

[0090] Preparation process: (1) Culture medium preparation: Same as in Example 1, except that the amount of DMEM basic and Advanced DMEM / F12 was not adjusted and no fibroblast supernatant was added.

[0091] (2) Organoid construction: Same as in Example 1.

[0092] (3) Organoid passage: Same as in Example 1.

[0093] Table 4. Cell counts in Example 3 and Comparative Example 6 ; Results and Analysis: As shown in Table 4 and Figure 4 As shown in Table 4, the cell counts for Example 3 and Comparative Example 6 are as follows. Figure 4 Bright-field micrographs of the cultured organoids from Example 3 and Comparative Example 6 are shown. For pancreatic cancer samples with less than 20% tumor parenchymal cell content, the culture results of Example 3 were significantly better than those of Comparative Example 6. Figure 4Bright-field micrographs show that organoids were successfully formed in Example 3. Despite the low initial tumor cell content, relatively complete and clearly morphologically distinct organoids were still visible. In contrast, almost no typical organoid structures were observed in Comparative Example 6, with only a few cell fragments or unformed cell clusters, indicating culture failure. The results indicate that when the initial sample has a low tumor cell content, using enhanced culture medium can effectively improve the success rate of organoid construction. Example 3, by adjusting the amounts of DMEM basic and Advanced DMEM / F12 and adding fibroblast supernatant, provided stronger support for tumor cell growth. The fibroblast supernatant may contain various growth factors and cytokines, which help simulate the in vivo tumor microenvironment and promote the survival and proliferation of a small number of tumor cells; while optimizing the basic culture medium ratio better meets the nutritional needs of tumor cells. Comparative Example 6, without these adjustments, could not effectively support the growth of low-content tumor cells, resulting in extremely poor culture performance. This comparison confirms that for samples with low tumor cell content, using enhanced culture medium strategies is key to improving the success rate of organoid culture.

[0094] Example 4

[0095] Sample source: Surgically removed pancreatic cancer tissue. Sequencing confirmed that the pancreatic cancer tissue was KRAS mutant and the tumor parenchymal cell content was less than 20%.

[0096] Preparation process: (1) Culture medium preparation: Based on the enhanced culture medium for pancreatic cancer organoids in Example 3, the concentrations of Wnt3a, RSPO1 and Noggin were adjusted to 0.1 μg / mL to prepare a complex culture medium for pancreatic cancer organoids.

[0097] (2) Organoid construction: Same as in Example 1.

[0098] (3) Organoid passage: Same as in Example 1.

[0099] Comparative Example 7 Sample source: Same as Example 4.

[0100] Preparation process: (1) Culture medium preparation: Same as in Example 1, except that the concentrations of Wnt3a, RSPO1 and Noggin were not adjusted, the amounts of DMEM basic and Advanced DMEM / F12 were not adjusted, and fibroblast supernatant was not added.

[0101] (2) Organoid construction: Same as in Example 1.

[0102] (3) Organoid passage: Same as in Example 1.

[0103] Table 5. Cell counts in Example 4 and Comparative Example 7 ; Results and Analysis: As shown in Table 5 and Figure 5 As shown in Table 5, the cell counts for Example 4 and Comparative Example 7 are as follows. Figure 5 Bright-field micrographs of the cultured organoids from Example 4 and Comparative Example 7 are shown. For pancreatic cancer samples with both KRAS mutations and tumor parenchymal cell content below 20%, the composite culture medium used in Example 4 showed better results than that in Comparative Example 7, with cell numbers of 7.75 × 10⁻⁶ cells. 4 / mL and 5.52×10 4 / mL. From Figure 5 As can be seen, Example 4 produced a larger number of organoids with relatively complete and clear structures, some exhibiting typical three-dimensional morphology. In contrast, while organoid formation was observed in Comparative Example 7, the number was smaller, and some organoids showed irregular shapes, blurred edges, or loose aggregation. The results indicate that, addressing the dual challenges of KRAS mutation and low tumor cell content, Example 4, by comprehensively adjusting the concentrations of Wnt3a, RSPO1, and Noggin, optimizing the basal culture medium ratio, and adding fibroblast supernatant, provided a more suitable growth microenvironment for tumor cells, significantly improving the efficiency and quality of organoid construction. Comparative Example 7, lacking the aforementioned comprehensive optimization, failed to fully meet the specific needs of this challenging sample, resulting in relatively poor culture performance.

[0104] Example 5

[0105] Sample source: surgically removed lung cancer tissue.

[0106] Preparation process: (1) Culture medium preparation: lung cancer organoid culture medium was prepared according to the organoid culture medium formula table shown in Table 1; (2) Organoid construction: Same as in Example 1.

[0107] (3) Organoid passage: Same as in Example 1.

[0108] Comparative Example 8 Sample source: surgically removed lung cancer tissue.

[0109] Preparation process: (1) Culture medium preparation: Same as comparative example 1.

[0110] (2) Organoid construction: Same as in Example 1.

[0111] (3) Organoid passage: Same as in Example 1.

[0112] Comparative Example 9 Sample source: surgically removed lung cancer tissue.

[0113] Preparation process: (1) Culture medium preparation: Unlike the lung cancer organoid culture medium in Example 5, N-2 additive was not added, but 5 μL of estradiol was added. Other components and amounts were the same as in Example 5.

[0114] (2) Organoid construction: Same as in Example 1.

[0115] (3) Organoid passage: Same as in Example 1.

[0116] Table 6. Cell counts in Example 5 and Comparative Examples 8-9 ; Results and Discussion: As shown in Table 6 and Figure 6 As shown in Table 6, the cell counts for Example 5 and Comparative Examples 8-9 are as follows. Figure 6 Bright-field micrographs of the cultured organoids from Example 5 and Comparative Examples 8-9 show that different culture medium components significantly affect the culture outcome of lung cancer organoids. The lung cancer organoid culture medium used in Example 5 exhibited the best culture results. Figure 6 As can be seen, the organoids cultured in Example 5 exhibited typical three-dimensional structures, with regular and plump morphologies, clear edges, and were numerous and uniform in size. In contrast, the results of Comparative Example 8 (universal culture medium) were extremely poor, with cell numbers far lower than in Example 5. This culture medium lacked key components such as B27, nicotinamide, fenestrated cinnamic acid, and dexamethasone, confirming the necessity of these components for the initial culture of lung cancer organoids. Comparative Example 9 added estradiol, but the culture results were still inferior to those of Example 5. Figure 6 As can be seen, although organoid formation was observed in Comparative Example 9, the number was significantly less than in Example 5, and some organoids exhibited irregular morphology and loose structure, suggesting that the additional addition of estradiol may have interfered with the optimized formulation of the culture medium, adversely affecting the normal development of organoids. In summary, the culture medium formulation of Example 5 has significant advantages in promoting the growth of lung cancer organoids and maintaining good morphology.

[0117] Example 6 Sample source: surgically removed lung cancer tissue, which was confirmed by sequencing to be KRAS mutant.

[0118] Preparation process: (1) Culture medium preparation: Based on the lung cancer organoid culture medium in Example 5, the concentrations of Wnt3a, RSPO1 and Noggin were adjusted to 0.1 μg / mL to prepare lung cancer KRAS mutant organoid culture medium.

[0119] (2) Organoid construction: Same as in Example 1.

[0120] (3) Organoid passage: Same as in Example 1.

[0121] Comparative Example 10 Sample source: Same as Example 6.

[0122] Preparation process: (1) Culture medium preparation: Same as in Example 5, except that the concentrations of Wnt3a, RSPO1 and Noggin were not adjusted compared to the lung cancer KRAS mutant organoid culture medium in Example 6.

[0123] (2) Organoid construction: Same as in Example 1.

[0124] (3) Organoid passage: Same as in Example 1.

[0125] Table 7 Cell counts in Example 6 and Comparative Example 10 ; Results and Discussion: As shown in Table 7 and Figure 7 As shown in Table 7, the cell counts for Example 6 and Comparative Example 10 are as follows. Figure 7 Bright-field micrographs of the cultured organoids from Example 6 and Comparative Example 10 show that adjusting the culture medium composition to target the KRAS mutation has a decisive impact on the culture effect of lung cancer organoids. The KRAS mutation-optimized medium used in Example 6 exhibited excellent culture results, yielding a cell count as high as 7.95 × 10⁻⁶. 5 / mL. From Figure 7 As can be seen, the organoids in Example 6 formed densely, exhibiting a typical three-dimensional structure with regular and full morphology, clear edges, and good growth. In contrast, the results of Comparative Example 10 were extremely poor, with only 7.5 × 10⁻⁶ cells. 3 / mL, which is two orders of magnitude different from Example 6. From Figure 7 As can be seen, almost no obvious organoid formation was observed in Comparative Example 10; only a small amount of cell debris or unformed cell clusters were observed, indicating that the culture process had essentially failed. In conclusion, optimizing the culture medium composition for KRAS-mutant lung cancer organoids can significantly improve the success rate of organoid construction, a strategy consistent with the findings of studies on pancreatic cancer organoids.

[0126] Example 7 Sample source: surgically removed lung cancer tissue with a tumor parenchymal cell content of less than 20%.

[0127] Preparation process: (1) Culture medium preparation: Based on the lung cancer organoid culture medium in Example 5, the addition amounts of DMEM basic and Advanced DMEM / F12 were adjusted to 20 mL and 13 mL respectively, and 15 mL of fibroblast supernatant was added to prepare lung cancer organoid enhanced culture medium.

[0128] (2) Organoid construction: Same as in Example 1.

[0129] (3) Organoid passage: Same as in Example 1.

[0130] Comparative Example 11 Sample source: Same as Example 7.

[0131] Preparation process: (1) Culture medium preparation: Same as in Example 5, except that the amount of DMEM basic and Advanced DMEM / F12 was not adjusted and no fibroblast supernatant was added, unlike the enhanced culture medium for pancreatic cancer organoids in Example 7.

[0132] (2) Organoid construction: Same as in Example 1.

[0133] (3) Organoid passage: Same as in Example 1.

[0134] Table 8. Cell counts in Example 7 and Comparative Example 11 ; Results and Discussion: As shown in Table 8 and Figure 8 As shown in Table 8, the cell counts for Example 7 and Comparative Example 11 are as follows. Figure 8 Bright-field micrographs of the cultured organoids from Example 7 and Comparative Example 11 show that, for lung cancer samples with low tumor parenchymal cell content, the enhanced culture medium strategy can significantly improve organoid culture results. The enhanced culture medium used for lung cancer organoids in Example 7 exhibited good culture results, yielding a cell count of 8.36 × 10⁻⁶. 4 / mL. From Figure 8 As can be seen, organoids were successfully formed in Example 7. Although the initial tumor cell content was low, relatively complete and clearly morphologically distinct organoids were still visible, with some exhibiting typical three-dimensional structures. In contrast, the results of Comparative Example 11 were extremely poor, with a cell count of only 2.25 × 10⁻⁶. 3 / mL, significantly lower than in Example 7. From Figure 8 As can be seen, almost no typical organoid structures were observed in Comparative Example 11, indicating that the culture process had essentially failed. For lung cancer samples with low tumor cell content, employing a culture medium enhancement strategy is key to improving the success rate of organoid culture, a finding consistent with research findings on pancreatic cancer organoids.

[0135] Example 8 Sample source: surgically removed lung cancer tissue. Sequencing confirmed that the lung cancer tissue was KRAS mutant and the tumor parenchymal cell content was less than 20%.

[0136] Preparation process: (1) Culture medium preparation: Based on the lung cancer organoid enhanced culture medium in Example 7, the concentrations of Wnt3a, RSPO1 and Noggin were adjusted to 0.1 μg / mL to prepare lung cancer organoid complex culture medium.

[0137] (2) Organoid construction: Same as in Example 1.

[0138] (3) Organoid passage: Same as in Example 1.

[0139] Comparative Example 12 Sample source: Same as Example 8.

[0140] Preparation process: (1) Culture medium preparation: Same as in Example 5, except that the concentrations of Wnt3a, RSPO1 and Noggin were not adjusted, the amounts of DMEM basic and Advanced DMEM / F12 were not adjusted, and fibroblast supernatant was not added.

[0141] (2) Organoid construction: Same as in Example 1.

[0142] (3) Organoid passage: Same as in Example 1.

[0143] Table 9. Cell numbers in Example 8 and Comparative Example 12 ; Results and Discussion: As shown in Table 9 and Figure 9 As shown in Table 9, the cell counts for Example 8 and Comparative Example 12 are as follows. Figure 9 Bright-field micrographs of the cultured organoids from Example 8 and Comparative Example 12 show that the composite culture medium strategy can significantly improve organoid culture results for lung cancer samples with both KRAS mutations and low tumor parenchymal cell content. The lung cancer organoid composite culture medium used in Example 8 exhibited excellent culture results, yielding a cell count of 9.61 × 10⁻⁶ cells. 5 / mL. From Figure 9 As can be seen, the organoids in Example 8 formed densely, exhibiting a typical three-dimensional structure with regular and full morphology, clear edges, good growth status, and numerous and relatively uniform in size. In contrast, the culture effect of Comparative Example 12 was significantly poor, with a cell count of only 6.13 × 10⁻⁶. 4 / mL, less than one-tenth of that in Example 8. From Figure 9As can be seen, although a small number of organoids were observed in Comparative Example 12, their number was significantly less than that in Example 8, and some organoids exhibited irregular morphology, loose structure, or blurred edges, indicating poor overall growth. This is because Comparative Example 12 did not undergo comprehensive optimization, failing to meet the signaling requirements of KRAS-mutant cells and providing sufficient growth support for low-content tumor cells, resulting in relatively poor culture outcomes. For lung cancer samples with both specific gene mutations and low cell content, employing a combined optimization strategy is crucial for improving the success rate of organoid culture, a finding highly consistent with research results on pancreatic cancer organoids.

[0144] Example 9 Sample source: Surgically removed breast cancer tissue.

[0145] Preparation process: (1) Culture medium preparation: Prepare breast cancer organoid culture medium according to the organoid culture medium formula table shown in Table 1; (2) Organoid construction: Same as in Example 1.

[0146] (3) Organoid passage: Same as in Example 1.

[0147] Comparative Example 13 Sample source: Surgically removed breast cancer tissue.

[0148] Preparation process: (1) Culture medium preparation: Same as comparative example 1.

[0149] (2) Organoid construction: Same as in Example 1.

[0150] (3) Organoid passage: Same as in Example 1.

[0151] Comparative Example 14 Sample source: Surgically removed breast cancer tissue.

[0152] Preparation process: (1) Culture medium preparation: Unlike the breast cancer organoid culture medium in Example 9, estradiol was not added, but 500 μL of N-2 additive was added. Other components and amounts were the same as in Example 9.

[0153] (2) Organoid construction: Same as in Example 1.

[0154] (3) Organoid passage: Same as in Example 1.

[0155] Table 10 Cell counts in Example 9 and Comparative Examples 13-14 ; Results and Discussion: As shown in Table 10 and Figure 10As shown in Table 10, the cell counts for Example 9 and Comparative Examples 13-14 are as follows. Figure 10 Bright-field micrographs of the cultured organoids from Example 9 and Comparative Examples 13-14 show that different culture medium components have a significant impact on the culture effect of breast cancer organoids. The breast cancer organoid culture medium used in Example 9 exhibited the best culture effect, yielding 2.73 × 10⁻⁶ cells. 5 / mL. From Figure 10 As seen in the bright-field micrographs, the organoids cultured in Example 9 exhibited typical three-dimensional structures with regular, plump morphologies, clear edges, numerous organoids of relatively uniform size, and good growth. In contrast, Comparative Example 13 showed extremely poor results, with a significantly lower cell count than Example 9. The lack of B27, nicotinamide, fenestrated cinnamic acid, and dexamethasone in the culture medium was the main reason for the extremely low culture efficiency of Comparative Example 13, confirming their necessity for initiating breast cancer organoid culture. Figure 10 As can be seen, almost no obvious organoid formation was observed in Comparative Example 13, with only a small amount of cell debris or unformed cell clusters. Comparative Example 14, which added N-2 additive, did not achieve ideal culture results, with a cell count of 1.86 × 10⁻⁶. 4 / mL. From Figure 10 As can be seen, although a small number of organoids were observed in Comparative Example 14, their number was significantly less than that in Example 9. Furthermore, some organoids exhibited irregular morphology, loose structure, or blurred edges, suggesting that the addition of N-2 may have disrupted the optimized culture medium formulation, interfering with the normal development of breast cancer organoids. In conclusion, the culture medium formulation of Example 9 demonstrates significant advantages in promoting the growth of breast cancer organoids and maintaining their favorable morphology, proving the necessity of optimizing culture medium components for different cancer types.

[0156] Example 10 Sample source: surgically removed breast cancer tissue with a tumor parenchymal cell content of less than 20%.

[0157] Preparation process: (1) Culture medium preparation: Based on the breast cancer organoid culture medium in Example 9, the addition amounts of DMEM basic and Advanced DMEM / F12 were adjusted to 20 mL and 13 mL respectively, and 15 mL of fibroblast supernatant was added to prepare breast cancer organoid enhanced culture medium.

[0158] (2) Organoid construction: Same as in Example 1.

[0159] (3) Organoid passage: Same as in Example 1.

[0160] Comparative Example 15 Sample source: Same as Example 9.

[0161] Preparation process: (1) Culture medium preparation: Same as in Example 9, except that the amount of DMEM basic and Advanced DMEM / F12 was not adjusted and no fibroblast supernatant was added, unlike the enhanced culture medium for breast cancer organoids in Example 10.

[0162] (2) Organoid construction: Same as in Example 1.

[0163] (3) Organoid passage: Same as in Example 1.

[0164] Table 11 Cell counts in Example 10 and Comparative Example 15 ; Results and Discussion: As shown in Table 11 and Figure 11 As shown in Table 11, the cell counts for Example 10 and Comparative Example 15 are as follows. Figure 11 Bright-field micrographs of the cultured organoids from Example 10 and Comparative Example 15 are shown. For breast cancer samples with less than 20% tumor parenchymal cell content, the culture effect of Example 10 was significantly better than that of Comparative Example 15, with cell numbers of 3.21 × 10⁻⁶ cells. 4 / mL and 0.73×10 3 / mL, the difference between the two is approximately 44 times. From Figure 11 As can be seen, organoids were successfully formed in Example 10. Despite the low initial tumor cell content, relatively complete and clearly morphologically distinct organoids were still observed, with some exhibiting typical three-dimensional structures. In contrast, almost no typical organoid structures were observed in Comparative Example 15, with only a small number of cell fragments or unformed cell clusters, indicating that the culture had essentially failed. The results show that when the tumor cell content in the starting sample is low, using enhanced culture medium can effectively improve the success rate of breast cancer organoid construction, which is consistent with the research results on pancreatic cancer and lung cancer organoids.

[0165] In summary, this invention constructs a dynamic and adaptable culture medium optimization method through a preparation process that includes conditional judgment and adjustment. Experimental results verify that this method can provide precise culture medium formulation solutions for the heterogeneity of tumor samples across three key dimensions: different types, gene mutation states, and cell content. This significantly improves the success rate and culture quality of various tumor organoids. Furthermore, the commercialized kit format simplifies the operation process, enhances experimental standardization and reproducibility, and provides reliable and efficient technical support for the subsequent clinical translation of tumor organoid technology.

[0166] The technical solution of the present invention has been described above with reference to the preferred embodiments shown in the accompanying drawings. However, it will be readily understood by those skilled in the art that the scope of protection of the present invention is obviously not limited to these specific embodiments. Without departing from the principles of the present invention, those skilled in the art can make equivalent changes or substitutions to the relevant technical features, and the technical solutions after these changes or substitutions will all fall within the scope of protection of the present invention.

[0167] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing an organoid culture medium, characterized in that, Includes the following steps: Step S1: Mix 50X serum-free B27, 10mM nicotinamide, 1.25mM N-acetylcysteine, Primocin, 10μMY-27632 2HCl, 0.05ng / mL Animal-Free KGF-2 / FGF-10, 10nM gastrin, 10μM SB202190, 10μM trichosine, 3μM dexamethasone and 1μM A8301 to obtain a first solution; Step S2: Add Wnt3a, RSPO1 and Noggin to the first solution, and based on the result that the tumor organoids are of the KRAS mutation type, increase the concentration of Wnt3a, RSPO1 and Noggin to obtain a second solution; Step S3: Based on the type of tumor organoid, determine whether to add N-2 additive or estradiol to the second solution to obtain a third solution; Step S4: Add DMEM basic and Advanced DMEM / F12 to the third solution to obtain organoid culture medium.

2. The method for preparing organoid culture medium according to claim 1, characterized in that, In step S1, the following amounts were added: 1 mL of 50X serum-free B27, 500 μL of 10 mM nicotinamide, 125 μL of 1.25 mM acetylcysteine, 100 μL of Primocin, 50 μL of 10 μM Y-276322HCl, 5 μL of 0.05 ng / mL Animal-Free KGF-2 / FGF-10, 5 μL of 10 nM gastrin, 5 μL of 10 μM SB202190, 50 μL of 10 μM trichosine, 50 μL of 3 μM dexamethasone, and 2 μL of 1 μM A8301.

3. The method for preparing organoid culture medium according to claim 2, characterized in that, In step S1, the concentration of Primocin in the first solution is 100 μg / mL.

4. The method for preparing organoid culture medium according to claim 3, characterized in that, In step S2, the concentrations of Wnt3a, RSPO1, and Noggin are all 0.05 μg / mL, and the addition amounts of Wnt3a, RSPO1, and Noggin are all 25 μL.

5. The method for preparing organoid culture medium according to claim 4, characterized in that, Step S2 further includes increasing the concentrations of Wnt3a, RSPO1, and Noggin to 0.1 μg / mL based on the result that the tumor organoid is a KRAS mutant.

6. The method for preparing organoid culture medium according to claim 5, characterized in that, Step S3 further includes, based on the result that the tumor organoid is a lung cancer organoid, determining to add 500 μL of N-2 additive to the second solution.

7. The method for preparing organoid culture medium according to claim 6, characterized in that, Step S3 further includes, based on the result that the tumor organoid is a breast cancer organoid, determining to add 5 μL of 5 μg / mL estradiol to the second solution.

8. The method for preparing organoid culture medium according to claim 7, characterized in that, Step S4 also includes adding 30 mL of DMEM basic and 18 mL of Advanced DMEM / F12.

9. The method for preparing organoid culture medium according to claim 8, characterized in that, Step S4 further includes adjusting the amount of DMEM basic added to 20 mL and the amount of Advanced DMEM / F12 added to 13 mL based on the result that the content of tumor parenchymal cells is lower than the preset content, and adding 15 mL of fibroblast supernatant to the third solution.

10. A kit containing an organoid culture medium prepared according to any one of claims 1-9, characterized in that, include: The organoid culture medium, rinsing solution, tissue digestion solution, neutralizing solution, and passage digestion solution are provided, wherein the rinsing solution contains bovine serum albumin and phosphate buffer, the tissue digestion solution contains a collagenase combination, the neutralizing solution contains basal culture medium, buffer, supplemented amino acids, and antibiotics, and the passage digestion solution contains protease and metal ion chelating agent.

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