A prostate cancer organoid special culture medium and a culture method thereof

Abstract

The application provides a prostate cancer organoid special culture medium and a culture method thereof, and belongs to the technical field of biological medicine. The prostate cancer organoid special culture medium is composed of a basic culture medium and a culture additive. The basic culture medium does not contain exogenous added Noggin recombinant protein and R-spondin recombinant protein. The culture additive comprises basic fibroblast growth factor, keratinocyte growth factor, fibroblast growth factor 10, insulin, p38MAPK signal pathway inhibitor, insulin-like growth factor 1, B27, nicotinamide, N-acetylcysteine, hydrocortisone, ROCK kinase inhibitor, retinoic acid, interleukin 6, prostaglandin E2, forskolin and androgen. The prostate cancer organoid special culture medium can reduce the cost and improve the culture success rate.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a culture medium for prostate cancer organoids and its culture method. Background Technology

[0002] Prostate cancer (PCa) is one of the most common malignant tumors in men worldwide. Its complex spatial, morphological, and genetic heterogeneity makes it difficult to treat, and the mechanisms of tumor transformation leading to clinical heterogeneity remain unclear. Therefore, there is an urgent need for preclinical models that can comprehensively reflect the heterogeneity of prostate cancer.

[0003] Prostate cancer research models mainly include traditional cell lines, conditionally reprogrammed cells (CRC), patient-derived xenograft models (PDX), genetically engineered mouse models (GEMM), and organoid models. Traditional cell lines (such as LNCaP and VCaP) are low-cost, easy to edit, and suitable for high-throughput screening, but lack tumor heterogeneity and microenvironment. CRC technology can achieve unlimited in vitro proliferation of primary cells, but has a weak androgen response and is easily contaminated by feeder cells. PDX models can highly simulate tumor heterogeneity and are related to clinical efficacy, but are costly, time-consuming, and difficult to use for high-throughput screening and gene editing. GEMM models have a complete microenvironment and immune system, but their construction is time-consuming and expensive, and they usually only simulate a few gene variations.

[0004] Organoid models have emerged as an ideal complementary approach due to their unique advantages: derived from pluripotent stem cells or organ progenitor cells, these models form organoid structures in three-dimensional culture, better preserving clinical tumor heterogeneity and supporting gene manipulation and high-throughput drug screening, while exhibiting high correlation with drug efficacy against the patient's primary tumor. Organoid models are an important cutting-edge tool for studying prostate cancer heterogeneity.

[0005] Despite significant progress in the culture of tumor organoids, limitations remain. The principle behind tumor organoid culture is to activate the Wnt / β-catenin pathway, thereby promoting cell proliferation in vitro. This pathway is crucial for embryonic stem cell development. While activation of this pathway promotes cell proliferation, it may also alter the stability of the tumor cell genome, thus changing the tumor's mutational profile. Researchers have demonstrated that after long-term passage culture, the tumor's gene mutational profile drifts compared to the original tumor. This drift may be related to various factors, including culture conditions, selective cell growth, and changes in the cellular microenvironment.

[0006] Existing technologies commonly add recombinant Wnt signaling-regulating protein R-Spondin or developmental regulatory protein Noggin to the main components of prostate cancer organoid culture media. For example, patent document CN116286654A discloses a prostate cancer organoid culture medium, comprising basal culture medium, basal cytokines, specific additive factors, inhibitors, and antibiotics; the basal cytokines include the following components at final concentrations: EGF, 10-100 ng / ml; Noggin, 20-500 ng / ml; R-spondin1, 20-500 ng / ml; Wnt3a, 20-500 ng / ml; FGF10, 1-50 ng / ml; FGF2, 1-50 ng / ml; the specific additive factors include the following components at final concentrations: Protodioscin, 0.1-50 nM; Dehydroepiandrosteroneacetate, 1-10 μM; SCF, 0.5-50 ng / mL. The core components used in this technology, Noggin, R-spondin 1, and Wnt3a, suffer from high costs, reliance on imports, and a fragile supply chain, which greatly hinders the large-scale culture and wider application of prostate cancer organoids.

[0007] Furthermore, prostate cancer organoid culture also suffers from low success rates and low passage efficiency. According to literature reports, the overall success rate of prostate cancer organoid culture is only 15%-20% (Gao D, Vela I, Sboner A, Iaquinta PJ, Karthaus WR, Gopalan A, et al. Organoid Cultures Derived From Patients With Advanced Prostate Cancer. Cell (2014) 159:176-87.), further limiting the widespread development of diverse clinical models. A study of 81 prostate cancer specimens with different pathological and clinical features (Servant R, Garioni M, Vlajnic T, Blind M, Pueschel H, Müller DC, et al. Prostate Cancer Patient-Derived Organoids: Detailed Outcome From a Prospective Cohort of 81 Clinical Specimens. J Pathol (2021) 254:543-55.) showed that the organoid success rate for metastatic prostatectomy specimens was 4 / 9, while the success rate for transurethral resection of the prostate (TURP) specimens was only 4 / 14.

[0008] Therefore, there is an urgent need to develop a low-cost, practical prostate cancer organoid culture medium that can better preserve the tumor gene mutation profile to meet current needs. Summary of the Invention

[0009] The technical problem to be solved by the present invention is to provide a special culture medium for prostate cancer organoids, which reduces costs and improves the success rate of culture, in order to address the shortcomings of the prior art.

[0010] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is: a special culture medium for prostate cancer organoids, which consists of a basal culture medium and culture additives. The basal culture medium does not contain exogenously added recombinant Noggin protein and recombinant R-spondin protein. The culture additives include basic fibroblast growth factor, keratinocyte growth factor, fibroblast growth factor 10, insulin, p38MAPK signaling pathway inhibitor, insulin-like growth factor 1, B27, nicotinamide, N-acetylcysteine, hydrocortisone, ROCK kinase inhibitor, retinoic acid, interleukin-6, prostaglandin E2, trichomoniasis, and androgens.

[0011] Optionally, the basal culture medium is Advanced DMEM / F12 medium, containing one or more of penicillin-streptomycin solution, HEPES buffer, GlutaMax and Primocin solution.

[0012] Optionally, the penicillin-streptomycin solution has a volume fraction of 1%, the HEPES buffer has a volume fraction of 1%, the GlutaMax has a volume fraction of 1%, and the Primocin solution has a volume fraction of 0.1%.

[0013] Optionally, the working concentration of the basic fibroblast growth factor is 10-50 μg / mL, and can be selected as 10 μg / mL, 20 μg / mL, 30 μg / mL, 40 μg / mL or 50 μg / mL;

[0014] The working concentration of the keratinocyte growth factor is 5-10 μg / mL, and can be selected as 5 μg / mL, 6 μg / mL, 7 μg / mL, 8 μg / mL, 9 μg / mL or 10 μg / mL;

[0015] The working concentration of the fibroblast growth factor 10 is 10-50 μg / mL, and can be selected as 10 μg / mL, 20 μg / mL, 30 μg / mL, 40 μg / mL or 50 μg / mL;

[0016] The working concentration of the insulin is 10-50 mg / mL, and can be selected as 10 mg / mL, 20 mg / mL, 30 mg / mL, 40 mg / mL or 50 mg / mL;

[0017] The working concentration of the p38MAPK signaling pathway inhibitor is 0.5-5 μM, and can be selected from 0.5, 0.7, 1, 2, 3, 4 or 5 μM;

[0018] The working concentration of insulin-like growth factor 1 is 50-100 ng / mL, and can be selected as 50, 60, 70, 80, 90 or 100 ng / mL;

[0019] The working concentration of B27 is 2%;

[0020] The working concentration of the nicotinamide is 5-50 μM, and can be selected as 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 μM;

[0021] The working concentration of N-acetylcysteine ​​is 100-200 μM, and can be selected as 100, 120, 130, 150, 170, 180 or 200 μM;

[0022] The working concentration of the hydrocortisone is 0.1-5 μg / mL, and can be selected as 0.1, 0.5, 1, 2, 3, 4 or 5 μg / mL;

[0023] The working concentration of the ROCK kinase inhibitor is 1-10 μM, and can be selected as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 μM;

[0024] The working concentration of the retinoic acid is 10-50 nM, and can be selected as 10, 20, 30, 40 or 50 nM;

[0025] The working concentration of interleukin-6 is 0.1-5 ng / mL, and can be selected as 0.1, 0.5, 1, 2, 3, 4 or 5 ng / mL;

[0026] The working concentration of the prostaglandin E2 is 0.5-5 μM, and can be selected as 0.5, 1, 2, 3, 4 or 5 μM;

[0027] The working concentration of the trichoin is 0.5-5 μM, and can be selected as 0.5, 1, 2, 3, 4 or 5 μM;

[0028] The working concentration of the androgen is 0.5-5 nM, and can be selected from 0.5, 0.8, 1, 2, 3, 4 or 5 nM.

[0029] Optionally, the p38MAPK signaling pathway inhibitor is SB202190, the ROCK kinase inhibitor is Y-27632, and the androgen is dihydrotestosterone.

[0030] Optionally, the culture additive consists of the following components at working concentrations: basic fibroblast growth factor: 10 μg / mL, keratinocyte growth factor: 5 μg / mL, fibroblast growth factor 10: 20 μg / mL, insulin: 10 mg / mL, SB202190: 0.5 μM, insulin-like growth factor 1: 50 ng / mL, B27: 2%, nicotinamide: 30 μM, N-acetylcysteine: 150 μM, hydrocortisone 0.5 μg / mL, Y-276321: 5 μM, retinoic acid: 25 nM, interleukin 6: 1 ng / mL, prostaglandin E2: 1 μM, trichoderma 1 μM, and dihydrotestosterone: 1 nM.

[0031] Optionally, the prostate cancer organoid culture medium is used to culture organoids obtained from tissue samples of prostate cancer patients.

[0032] A method for preparing the above-mentioned prostate cancer organoid culture medium includes: first preparing a basic culture medium, then adding culture additives according to the above-mentioned content, and mixing evenly to obtain the culture medium.

[0033] A prostate cancer organoid, obtained by culturing the prostate cancer organoid using a special culture medium.

[0034] A method for preparing the above-mentioned prostate cancer organoid includes the following steps:

[0035] (1) Cut the prostate cancer tissue sample into small pieces, wash it, and then add collagenase for digestion;

[0036] (2) After digestion, the cells were resuspended in cold Cultrex growth factor reduced BME type 2, inoculated into a cell culture plate, and placed in a cell culture incubator to solidify the BME containing the cells.

[0037] (3) Add the prostate cancer organoid culture medium to the cell culture plate, and culture it in a cell culture incubator to obtain prostate cancer organoids.

[0038] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0039] This invention completely abandons the expensive Wnt signaling-regulating recombinant protein R-spondin and the development-regulating recombinant protein Noggin. Through formulation optimization and combination, it simulates the microenvironment required for the growth of prostate cancer organoids and develops a new culture medium suitable for the growth of prostate cancer organoids.

[0040] This invention's specialized culture medium comprehensively considers various factors required for cell proliferation, differentiation, and functional maintenance, making it suitable for long-term culture and research of prostate cancer organoids. In this invention, basic fibroblast growth factor, keratinocyte growth factor, fibroblast growth factor 10, insulin-like growth factor 1, and insulin synergistically construct a suitable microenvironment for prostate cancer cell proliferation, driving cells into the cell cycle and undergoing division from different angles. B202190, sulphine, prostaglandin E2, and nicotinamide synergistically replace the function of Noggin / R-spondin, maintaining the stem / undifferentiated state. SB202190, in synergy with sulphine or prostaglandin E2, creates an environment for stem cell / progenitor cell self-renewal by inhibiting differentiation pathways and activating stem cell-promoting pathways. Nicotinamide further consolidates the undifferentiated state by inhibiting differentiation enzymes. Y-27632, N-acetylcysteine, B27, and hydrocortisone synergistically ensure cell survival and stability. Y-27632 prevents apoptosis in the early stages; the antioxidant components in N-acetylcysteine ​​and B27 work together to combat oxidative damage, reduce DNA oxidative damage, lower mutation rates, and improve genome stability; hydrocortisone provides signals to resist stress and promote survival. Dihydrotestosterone, keratinocyte growth factor, and fibroblast growth factor 10 synergistically provide tissue-specific signals. Dihydrotestosterone is used to ensure that the culture medium selectively supports the growth of prostate cancer cells dependent on AR signals, maintain tumor characteristics, and improve culture success rate. Keratinocyte growth factor and fibroblast growth factor 10 are important factors in prostate development and homeostasis. Retinoic acid is used in this invention to regulate the balance between differentiation and proliferation. Under the basis of proliferative signals, low concentrations of retinoic acid are used to induce mild and controllable differentiation, prevent excessive dense growth of organoids leading to internal cell death, and maintain their healthy state in long-term culture.

[0041] Based on this, the prostate cancer organoid culture medium of this invention, through the combination of SB202190, spiculin, PGE2, etc., eliminates the dependence on Noggin and R-spondin, solving the problems of high cost and import dependence of current culture media; through the antioxidant system composed of N-acetylcysteine ​​and B27, combined with the anti-apoptotic factor Y-27632, it significantly reduces cellular stress during culture and improves genomic stability; through the combination of dihydrotestosterone with keratinocyte growth factor and fibroblast growth factor 10, it provides a highly specific survival environment for prostate cancer cells and improves the success rate of culture; the culture medium components work together as a whole to construct a microenvironment that supports the stable growth of prostate cancer organoids in the absence of Wnt / BMP recombinant proteins.

[0042] The special culture medium of this invention provides better support and promotion for the growth of prostate cancer organoids, and typical prostate cancer organoids can be obtained in 7-16 days. It better preserves the consistency and heterogeneity of patient-derived tumors in vitro, and better maintains the stability of the tumor cell genome in vitro, laying the foundation for further research and application.

[0043] The cost of the special culture medium of this invention is significantly lower than that of other methods. The main reason is that it does not contain expensive recombinant proteins such as R-Spondin, which regulates Wnt signaling, or Noggin, which regulates development. Instead, it uses other specific components, which makes it possible to carry out large-scale prostate cancer organoid culture for drug screening and other research applications.

[0044] The application method of the special culture medium of this invention is clear and simple, with minimal impact on the culture results from the operator, thus improving the consistency of the culture results and providing convenient conditions for subsequent large-scale culture.

[0045] Using the special culture medium of this invention, prostate cancer organoids were cultured. After 7-16 days of growth, the diameter of the prostate cancer organoids grew to 50-200 μm, and the size was uniform. The resulting organoid masses grew well, had clear boundaries, and could be stably passaged for more than 10 generations.

[0046] The cell proliferation rate was confirmed to be approximately 95% by Ki67 staining, a marker of cell proliferation, and the apoptosis rate was confirmed to be less than 5% by cleaved caspase-3 staining, a marker of cell apoptosis.

[0047] The success rate of organoid culture for transurethral resection of the prostate (TURP) specimens is approximately 50%, which is more than 25% higher than that of existing technologies; the success rate of organoid culture for prostate cancer surgical specimens is more than 70%, which is more than 30% higher than that of existing technologies. Attached Figure Description

[0048] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0049] Figure 1 Microscopic images of organoid culture from a patient's prostate cancer surgical sample on days 1, 4, 8, 12, and 16.

[0050] Figure 2 Optical microscope image of prostate cancer organoid culture after long-term passage to day 98, 10th generation;

[0051] Figure 3 Circos maps of RNA sequencing genomes from prostate cancer organoids (PDOs) and prostate cancer tumor tissues (Patients) from the same patient. Detailed Implementation

[0052] To better understand the present invention, the following embodiments further illustrate the content of the invention, but the scope of protection of the present invention is not limited to the following embodiments. Numerous specific details are set forth in the following description to provide a more thorough understanding of the invention. However, it will be apparent to those skilled in the art that the present invention can be practiced without one or more of these details.

[0053] Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without inventive effort are within the scope of protection of this invention.

[0054] Unless otherwise specified, all raw materials are derived from commercially available products and do not contain any unspecified components other than unavoidable impurities.

[0055] In the embodiments of this invention, "%" represents the volume percentage of the component in the corresponding culture medium.

[0056] Example 1: A culture medium specifically for prostate cancer organoids, consisting of a basal culture medium and culture additives, wherein the basal culture medium is Advanced... DMEM / F12 medium, excluding exogenously added recombinant Noggin and R-spondin, contains 1% penicillin-streptomycin solution, 1% HEPES buffer, 1% GlutaMax, and 0.1% Primocin. Culture additives consist of the following components at working concentrations: basic fibroblast growth factor: 10 μg / mL, keratinocyte growth factor: 5 μg / mL, fibroblast growth factor 10: 20 μg / mL, insulin: 10 mg / mL, SB202190: 0.5 μM, insulin-like growth factor 1: 50 ng / mL, B27: 2%, nicotinamide: 30 μM, N-acetylcysteine: 150 μM, hydrocortisone: 0.5 μg / mL, Y-276321: 5 μM, retinoic acid: 25 nM, interleukin-6: 1 ng / mL, prostaglandin E2: 1 μM, trichosamine: 1 μM, and dihydrotestosterone: 1 nM.

[0057] Example 2: A culture medium specifically for prostate cancer organoids, differing from Example 1 in that the culture additives consist of the following components at working concentrations: basic fibroblast growth factor: 20 μg / mL, keratinocyte growth factor: 10 μg / mL, fibroblast growth factor 10: 50 μg / mL, insulin: 20 mg / mL, SB202190: 2 μM, insulin-like growth factor 1: 100 ng / mL, B27: 2%, nicotinamide: 5 μM, N-acetylcysteine: 200 μM, hydrocortisone: 0.1 μg / mL, Y-276321: 1 μM, retinoic acid: 10 nM, interleukin-6: 5 ng / mL, prostaglandin E2: 0.5 μM, trichosamine: 0.5 μM, and dihydrotestosterone: 0.5 nM.

[0058] Example 3: A culture medium specifically for prostate cancer organoids, differing from Example 1 in that the culture additives consist of the following components at working concentrations: basic fibroblast growth factor: 50 μg / mL, keratinocyte growth factor: 8 μg / mL, fibroblast growth factor 10: 10 μg / mL, insulin: 50 mg / mL, SB202190: 5 μM, insulin-like growth factor 1: 70 ng / mL, B27: 2%, nicotinamide: 50 μM, N-acetylcysteine: 100 μM, hydrocortisone: 5 μg / mL, Y-276321: 10 μM, retinoic acid: 50 nM, interleukin-6: 0.1 ng / mL, prostaglandin E2: 5 μM, trichomoniasis 5 μM, and dihydrotestosterone: 5 nM.

[0059] Example 4: A culture medium specifically for prostate cancer organoids, differing from Example 1 in that the culture additives consist of the following components at working concentrations: basic fibroblast growth factor: 30 μg / mL, keratinocyte growth factor: 6 μg / mL, fibroblast growth factor 10: 30 μg / mL, insulin: 30 mg / mL, SB202190: 1 μM, insulin-like growth factor 1: 85 ng / mL, B27: 2%, nicotinamide: 20 μM, N-acetylcysteine: 120 μM, hydrocortisone: 2 μg / mL, Y-276321: 3 μM, retinoic acid: 40 nM, interleukin-6: 3 ng / mL, prostaglandin E2: 3 μM, trichomoniasis 3 μM, and dihydrotestosterone: 3 nM.

[0060] Example 5: A culture medium specifically for prostate cancer organoids, differing from Example 1 in that the culture additives consist of the following components at working concentrations: basic fibroblast growth factor: 40 μg / mL, keratinocyte growth factor: 7 μg / mL, fibroblast growth factor 10: 40 μg / mL, insulin: 40 mg / mL, SB202190: 3 μM, insulin-like growth factor 1: 60 ng / mL, B27: 2%, nicotinamide: 40 μM, N-acetylcysteine: 170 μM, hydrocortisone: 3 μg / mL, Y-276321: 7 μM, retinoic acid: 30 nM, interleukin-6: 2 ng / mL, prostaglandin E2: 4 μM, trichomoniasis 2 μM, and dihydrotestosterone: 4 nM.

[0061] The prostate cancer organoid culture medium prepared in Examples 1-5 was used to culture organoids obtained from tissue samples of prostate cancer patients.

[0062] The preparation method of any of the prostate cancer organoid culture media prepared in Examples 1-5 includes: first preparing a basic culture medium, then adding culture additives in the above-mentioned amounts in sequence, mixing evenly, and thus obtaining the culture medium.

[0063] A prostate cancer organoid was obtained by culturing it using any of the prostate cancer organoid-specific culture media prepared in Examples 1-5.

[0064] Example 6: A method for preparing prostate cancer organoids, comprising the following steps:

[0065] (1) Cut the surgical tissue sample of the patient’s prostate cancer into small pieces of 0.5mm×0.5mm, wash them and add collagenase to digest for 60 minutes;

[0066] (2) Digest the cells according to 2×10 6 The cells were resuspended at a concentration of 1 cell / mL in cold Cultrex growth factor reduced BME type 2, inoculated into 24-well cell culture plates, 40 μL per well, and placed in a 37°C cell culture incubator for 20 minutes to solidify the BME containing the cells.

[0067] (3) Add 400 μL of the special culture medium prepared in Example 1 to each well, and culture in a cell culture incubator with 5% CO2 concentration and 37°C. Replace the special culture medium every 4 days, and obtain prostate cancer organoids in 16 days.

[0068] Optical micrographs of organoid cultures from prostate cancer surgical samples taken by the patient on days 1, 4, 8, 12, and 16 are shown below. Figure 1 As shown.

[0069] Figure 1The results showed that the diameter of the prostate cancer organoids was greater than 100 μm after 16 days of growth, and the resulting organoid masses grew well with clear boundaries and uniform size.

[0070] Optical microscope images of prostate cancer organoids cultured for 98 days (10th passage) are shown below. Figure 2 As shown, 2a is a 4x objective lens and 2b is a 20x objective lens.

[0071] Figure 2 The results showed that after 98 days of culture and passage to the 10th generation, the prostate cancer organoids still exhibited good condition, indicating that the prostate cancer organoids of this invention have the ability to be cultured and passaged for a long time.

[0072] Once the prostate cancer organoids grew to a diameter of 100-150 micrometers, they were digested with TrypLE Express enzyme, centrifuged, and the cell clumps were retained. RNA was extracted using Thermo Fisher Scientific's RNA extraction kit and sent to a sequencing company for whole genome and transcriptome sequencing.

[0073] RNA sequencing genome compilation Circos maps of prostate cancer organoids (PDOs) and prostate cancer tumor tissues from the same patient are shown below. Figure 3 As shown.

[0074] Figure 3 The results showed that patient-derived prostate cancer organoids (PDOs) and prostate cancer tumor tissues exhibited a high degree of genomic similarity, indicating that the PDO culture system of this invention effectively maintained the genomic stability of tumor cells during in vitro expansion.

[0075] The proportion of organoids that were successfully formed and stably expanded was statistically analyzed, and the results showed that the success rate of culture of prostate cancer organoid surgical specimens in this embodiment was approximately 70%.

[0076] Example 7: A method for preparing prostate cancer organoids, which differs from Example 6 in that: (3) 400 μL of the special culture medium prepared in Example 2 is added to each well, and the well is placed in a cell culture incubator with a 5% CO2 concentration and a temperature of 37°C. The special culture medium is replaced every 4 days, and prostate cancer organoids are obtained in 12 days.

[0077] Example 8: A method for preparing prostate cancer organoids, which differs from Example 6 in that: (3) 400 μL of the special culture medium prepared in Example 3 is added to each well, and the well is placed in a cell culture incubator with a 5% CO2 concentration and a temperature of 37°C. The special culture medium is replaced every 4 days, and prostate cancer organoids are obtained after 14 days.

[0078] Example 9: A method for preparing prostate cancer organoids, comprising the following steps:

[0079] (1) Cut the tissue sample from the transurethral resection of the prostate into small pieces of 0.5mm×0.5mm, wash and then add collagenase to digest for 60 minutes;

[0080] (2) Digest the cells according to 2×10 6 The cells were resuspended at a concentration of 1 cell / mL in cold Cultrex growth factor reduced BME type 2, inoculated into 24-well cell culture plates, 40 μL per well, and placed in a 37°C cell culture incubator for 20 minutes to solidify the BME containing the cells.

[0081] (3) Add 400 μL of the special culture medium prepared in Example 1 to each well, and culture in a cell culture incubator with 5% CO2 concentration and 37°C. Replace the special culture medium every 6 days, and obtain prostate cancer organoids in 14 days.

[0082] The success rate of culture of transurethral resection of the prostate specimen in this embodiment is approximately 50%.

[0083] The following are comparative examples.

[0084] Comparative Example 1: A prostate cancer organoid culture medium (refer to CN116286654 A), comprising basal medium, basal cytokines, specific additive factors, inhibitors, and antibiotics; the basal medium was DMEM / F12, and the basal cytokines included the following components at final concentrations: EGF, 30 ng / mL; Noggin, 200 ng / mL; R-spondin 1, 200 ng / mL; Wnt3a, 100 ng / mL; FGF10, 50 ng / mL; FGF2, 50 ng / mL; the specific additive factors included the following components at final concentrations: Protodioscin, 10 nM; Dehydroepiandrosterone Acetate (dehydroepiandrosterone acetate), 5 μM; SCF (stem cell factor), 5 ng / mL; inhibitors include the following components at final concentrations: SB202190, 400 nM; A83-01, 2 μM; Y-27632 dihydrochloride, 10 μM; antibiotics are primary cell antibiotics at a final concentration of 500 μg / mL.

[0085] Comparative Example 2: Unlike Example 1, only the basal medium was used. The basal medium was Advanced DMEM / F12 medium, which did not contain exogenously added Noggin recombinant protein and R-spondin recombinant protein. It contained 1% penicillin-streptomycin solution, 1% HEPES buffer, 1% GlutaMax and 0.1% Primocin, and no culture additives were added.

[0086] Comparative Example 3: Unlike Example 1, only SB202190 is omitted.

[0087] Comparative Example 4: Unlike Example 1, only nicotinamide was omitted.

[0088] Comparative Example 5: Unlike Example 1, only the tuftin was omitted.

[0089] Comparative Example 6: Unlike Example 1, only prostaglandin E2 was omitted.

[0090] Comparative Example 7: Unlike Example 1, SB202190, nicotinamide, salivain and prostaglandin E2 were omitted.

[0091] The culture media prepared in Comparative Examples 1-7 were used to prepare prostate cancer organoids using the method shown in Example 6.

[0092] The following experiments were conducted on the prostate cancer organoids prepared in Examples 6-8 and Comparative Examples 1-7:

[0093] 1. Statistical analysis of organoid culture success rate: Organoid culture success rate / % = (Number of cells forming organoids / Initial number of inoculated cells) × 100%. The results are shown in Table 1.

[0094] 2. Cell proliferation rate and apoptosis rate: Ki67 and cleaved caspase-3 fluorescence staining and quantification assays were performed. Specifically, after prostate cancer organoids grew to a diameter of 100 μm, the organoids were fixed with 4% paraformaldehyde. Then, primary antibodies against Ki67 (rabbit host) and cleaved caspase-3 (mouse host) were added, followed by rabbit secondary antibody (binding to green fluorescent protein), mouse secondary antibody (binding to red fluorescent protein), and the nuclear dye 4',6-diamidinyl-2-phenylindole (DAPI). Twenty random fields of view were photographed under a fluorescence microscope. The ratios of green (Ki67) to DAPI and red (cleaved caspase-3) to DAPI were calculated, and the cell proliferation rate and apoptosis rate were expressed as percentages. These two indicators reflect cell proliferation (Ki67) and apoptosis (cleaved caspase-3), respectively. The results are shown in Table 2.

[0095] Table 1. Statistical results of organoid culture success rate (unit: %)

[0096]

[0097] Table 1 shows that the success rate of prostate cancer organoid culture of the present invention is about 70%, which is better than that of the existing similar culture medium using a combination of Noggin, R-spondin 1 and Wnt3a (Comparative Example 1). This indicates that the present invention can reduce costs by more than 50% while maintaining culture efficiency comparable to conventional methods. Meanwhile, the results of Comparative Examples 2-7 show that both the basal culture medium and culture additives have a significant impact on the success rate of organoid culture.

[0098] Table 2 Results of cell proliferation rate and apoptosis rate

[0099]

[0100] The results in Table 2 show that the cell proliferation rate of the prostate cancer organoids of the present invention is about 95.0%, and the apoptosis rate is less than 5.0%, which is better than that of comparative examples 1-7. This indicates that the present invention abandons expensive recombinant proteins and provides a microenvironment conducive to the stable growth of prostate cancer cells based on component synergy.

[0101] In summary, the prostate cancer organoid culture medium of this invention is low in cost, high in culture efficiency, has excellent cell proliferation rate and low apoptosis rate, good genomic stability, and is suitable for long-term passage and application.

[0102] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Any other modifications or equivalent substitutions made by those skilled in the art to the technical solutions of the present invention, as long as they do not depart from the spirit and scope of the technical solutions of the present invention, should be covered within the scope of the claims of the present invention.

Claims

1. A culture medium specifically for prostate cancer organoids, characterized in that: The medium consists of a basal culture medium and culture supplements. The basal culture medium is Advanced DMEM / F12 medium containing 1% (v / v) penicillin-streptomycin solution, 1% (v / v) HEPES buffer, 1% (v / v) GlutaMax, and 0.1% (v / v) Primocin solution, and does not contain exogenously added Noggin recombinant protein and R-spondin recombinant protein. The culture supplements consist of the following components at working concentrations: basic fibroblast growth factor: 10-50 μg / mL, keratinocyte growth factor: 5-10 μg / mL, and fibroblast growth factor 10: 10-50 μg / mL. g / mL, Insulin: 10-50 mg / mL, SB202190: 0.5-5 μM, Insulin-like growth factor 1: 50-100 ng / mL, B27: 2%, Nicotinamide: 5-50 μM, N-acetylcysteine: 100-200 μM, Hydrocortisone 0.1-5 μg / mL, Y-27632: 1-10 μM, Retinoic acid: 10-50 nM, Interleukin-6: 0.1-5 ng / mL, Prostaglandin E2: 0.5-5 μM, Trichoderma 0.5-5 μM, Dihydrotestosterone: 0.5-5 nM.

2. The culture medium for prostate cancer organoids as described in claim 1, characterized in that: The culture additive consists of the following components at working concentrations: basic fibroblast growth factor: 10 μg / mL, keratinocyte growth factor: 5 μg / mL, fibroblast growth factor 10: 20 μg / mL, insulin: 10 mg / mL, SB202190: 0.5 μM, insulin-like growth factor 1: 50 ng / mL, B27: 2%, nicotinamide: 30 μM, N-acetylcysteine: 150 μM, hydrocortisone 0.5 μg / mL, Y-27632: 5 μM, retinoic acid: 25 nM, interleukin 6: 1 ng / mL, prostaglandin E2: 1 μM, trichoderma 1 μM, and dihydrotestosterone: 1 nM.

3. The use of the prostate cancer organoid culture medium as described in claim 1 or 2 for culturing organoids obtained from tissue samples of prostate cancer patients.

4. A method for preparing prostate cancer organoids using a special culture medium for prostate cancer organoids as described in claim 1 or 2, characterized in that: Includes the following steps: (1) Cut the prostate cancer tissue sample into small pieces, wash it, and then add collagenase for digestion; (2) After digestion, the cells were resuspended in cold Cultrex growth factor reduced BME type 2, inoculated into a cell culture plate, and placed in a cell culture incubator to solidify the BME containing the cells. (3) Add the prostate cancer organoid culture medium to the cell culture plate, and culture it in a cell culture incubator to obtain prostate cancer organoids.

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