Special culture medium for breast cancer organoid and culture method thereof

Abstract

The invention provides a special culture medium for breast cancer organoid and a culture method thereof, and belongs to the technical field of primary cell culture. The special culture medium for the breast cancer organoid is composed of a basic culture medium and a culture additive, and the basic culture medium is a DMEM (Dulbecco Modified Eagle Medium) / F12 culture medium. The culture additive is prepared from an epidermal growth factor, a basic fibroblast growth factor, a keratinocyte growth factor, a fibroblast growth factor 10, insulin, transferrin, sodium selenite, A83-01, SB202190, an insulin-like growth factor 1, B27, nicotinamide and N-acetylcysteine. According to the culture medium, the culture cost is reduced, and meanwhile, the stability of tumor cell genomes in the culture process is improved.

Description

Technical Field

[0001] The invention belongs to the technical field of primary cell culture, and specifically relates to a special culture medium for breast cancer organoids and a culture method thereof. Background Art

[0002] As human life expectancy increases and lifestyles change, the incidence and mortality of cancer are increasing year by year. Among women, the incidence of breast cancer is the highest and is on the rise, posing a serious threat to women's health. Breast cancer is a tumor that originates from mammary epithelial cells. Pathologists have long recognized that breast cancer has a high degree of tumor heterogeneity. One manifestation of tumor heterogeneity is that there are great differences between different tumors. Based on this, breast cancer is divided into different types to guide clinical medication and judge prognosis.

[0003] In recent years, the treatment of tumors has shifted from one treatment for all tumors to personalized precision treatment for tumor patients. Now, molecular biology methods are usually used to evaluate patients and select drugs. For example, in breast cancer, HER2 overexpression can be treated with targeted therapeutic drugs such as Herceptin, which greatly improves the survival rate of patients. However, the effectiveness of treatment is usually not determined by a single gene marker. Even the same marker has different efficacy in different tumors. For example, HER2 overexpression is a good therapeutic marker in breast cancer, but the same HER2 overexpression has little effect on digestive tract tumors. In this way, the in vitro culture of tumors plays an important role in predicting tumor treatment.

[0004] Over the past few decades, researchers have developed different culture methods to strive to most accurately reflect the in vivo status of tumors. Organoid culture is a culture method that cultures cells or tissues in vitro to form a 3D structure that is similar to the cell or tissue source. The earliest culture was of colon cells. Normal colon stem cells can be cultured and differentiated into the villus-like structure of the colon in vitro, while maintaining the stability of the genome. Soon this technology was used to culture colon cancer cells, and was extended to pancreatic cancer, liver cancer, prostate cancer, gastric cancer, etc.

[0005] Sachs et al. (Sachs N, de Ligt J, Kopper O, et al. A living biobank of breast cancer organoids captures disease heterogeneity[J]. Cell, 2018, 172(1 / 2): 373-386. e10.) successfully cultured breast cancer organoids. This patient-derived tumor in vitro model can accurately simulate the biological characteristics of tumors in vivo and their response to drug treatment, which is of great value for tumor research, drug testing and screening.

[0006] Although great progress has been made in the cultivation of tumor organoids, their cultivation methods still have their limitations. The principle of tumor organoid cultivation is usually to activate the Wnt / ß-catenin pathway of cells, so that cells can proliferate in vitro. This pathway is an important pathway for the development of embryonic stem cells. Although the activation of this pathway can promote cell proliferation, it may also change the stability of the tumor cell genome, thereby changing the mutation map of the tumor. Researchers have confirmed that after long-term subculture, the gene mutation map of the tumor has drifted compared with the original tumor. This drift of gene mutation may be related to a variety of factors, including culture conditions, selective growth of cells, and changes in the cell microenvironment.

[0007] In addition, it is generally necessary to add Wnt signaling regulating recombinant protein R-Spondin or developmental regulatory protein Noggin to the main components of breast cancer organoid culture medium. For example, patent document CN 111500540A discloses a breast cancer organoid culture kit, which contains Noggin, one or more of the Wnt signaling pathway-related secretory protein family R-Spondin1~4. Such recombinant proteins are expensive, and the main manufacturers are all located abroad, which leads to the high production cost of breast cancer organoid-specific culture medium, the need to import the main added ingredients, the long procurement cycle, and the supply cycle is easily affected by the external environment. These problems have greatly hindered the large-scale culture of breast cancer organoids and a wider range of application research.

[0008] Furthermore, breast cancer organoid culture still has problems such as low culture success rate and low passaging efficiency. Existing studies (Drost J, Clevers H. Organoids in cancer research[J]. Nat Rev Cancer, 2018,18(7): 407-418.) reported that the culture success rate of breast cancer organoids was about 60%. Sachs et al. (Sachs N, deLigt J, Kopper O, et al. A living biobank of breast cancer organoids captures disease heterogeneity[J]. Cell, 2018, 172(1 / 2): 373-386. e10.) reported that the culture success rate of breast cancer organoids could reach 80%, but the culture and passaging methods are cumbersome and the culture cost is high, which limits its promotion and application.

[0009] Therefore, there is an urgent need to develop a lower-cost, easier-to-use breast cancer organoid culture medium that is more capable of maintaining tumor gene mutation profiles. At the same time, in the field of scientific research, the culture of normal breast organoids will provide a solid foundation for studying the pathogenesis of breast cancer, so the development of a culture medium for normal breast organoids is also urgent. Summary of the invention

[0010] In view of the deficiencies of the prior art, the technical problem to be solved by the present invention is to provide a special culture medium for breast cancer organoids, which can reduce the culture cost while improving the stability of the tumor cell genome and the culture success rate during the culture process.

[0011] In order to solve the above technical problems, the technical solution adopted by the present invention is: A special culture medium for breast cancer organoids, consisting of a basal culture medium and a culture additive, wherein the basal culture medium is a DMEM / F12 culture medium, and the culture additive consists of epidermal growth factor, basic fibroblast growth factor, keratinocyte growth factor, fibroblast growth factor 10, insulin, transferrin, sodium selenite, A83-01, SB202190, insulin-like growth factor 1, B27, nicotinamide and N-acetylcysteine.

[0012] Optionally, the DMEM / F12 culture medium contains 1% penicillin-streptomycin, 1% HEPES buffer and 1% GlutaMax solution.

[0013] Optionally, the working concentration of the epidermal growth factor is 1-10 μg / mL, optionally 1 μg / mL, 2 μg / mL, 3 μg / mL, 4 μg / mL, 5 μg / mL, 6 μg / mL, 7 μg / mL, 8 μg / mL, 9 μg / mL or 10 μg / mL, preferably 3-8 μg / mL; The working concentration of the basic fibroblast growth factor is 10-50 μg / mL, and can be 10 μg / mL, 15 μg / mL, 20 μg / mL, 25 μg / mL, 30 μg / mL, 35 μg / mL, 40 μg / mL, 45 μg / mL or 50 μg / mL, preferably 10-30 μg / mL; The working concentration of the keratinocyte growth factor is 5-10 μg / mL, and can be 5 μg / mL, 6 μg / mL, 7 μg / mL, 8 μg / mL, 9 μg / mL or 10 μg / mL, preferably 5-8 μg / mL; The working concentration of the fibroblast growth factor 10 is 10-50 μg / mL, and can be 10 μg / mL, 15 μg / mL, 20 μg / mL, 25 μg / mL, 30 μg / mL, 35 μg / mL, 40 μg / mL, 45 μg / mL or 50 μg / mL, preferably 10-30 μg / mL; The working concentration of the insulin is 10-50 mg / mL, which can be 10 mg / mL, 15 mg / mL, 20 mg / mL, 25 mg / mL, 30 mg / mL, 35 mg / mL, 40 mg / mL, 45 mg / mL or 50 mg / mL, preferably 10-30 mg / mL; The working concentration of transferrin is 250-750 mg / L, and can be 250 mg / L, 300 mg / L, 350 mg / L, 400 mg / L, 450 mg / L, 500 mg / L, 550 mg / L, 600 mg / L, 650 mg / L, 700 mg / L or 750 mg / L, preferably 400-650 mg / L; The working concentration of sodium selenite is 0.5-1.0 mg / L, and can be 0.5 mg / L, 0.6 mg / L, 0.7 mg / L, 0.8 mg / L, 0.9 mg / L or 1.0 mg / L, preferably 0.5-0.8 mg / L; The working concentration of A83-01 is 1-10 mM, optionally 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM or 10 mM, preferably 5-10 mM; The working concentration of SB202190 is 1-10 mM, optionally 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM or 10 mM, preferably 5-10 mM; The working concentration of the insulin-like growth factor 1 is 50-100 ng / mL, optionally 50 ng / mL, 60 ng / mL, 70 ng / mL, 80 ng / mL, 90 ng / mL or 100 ng / mL, preferably 75-100 ng / mL; The working concentration of B27 is 2%; The working concentration of nicotinamide is 5-50 μM, which can be 5 μM, 10 μM, 15 μM, 20 μM, 25 μM, 30 μM, 35 μM, 40 μM, 45 μM or 50 μM, preferably 15-40 μM; The working concentration of the N-acetylcysteine ​​is 100-200 μM, and can be 100 μM, 110 μM, 120 μM, 130 μM, 140 μM, 150 μM, 160 μM, 170 μM, 180 μM, 190 μM or 200 μM, preferably 120-180 μM.

[0014] Preferably, the culture additive consists of the following working concentrations of ingredients: epidermal growth factor: 5 μg / mL, basic fibroblast growth factor: 10 μg / mL, keratinocyte growth factor: 5 μg / mL, fibroblast growth factor 10: 20 μg / mL, insulin: 10 mg / mL, transferrin: 550 mg / L, sodium selenite: 0.67 mg / L, A83-01: 10 mM, SB202190: 10 mM, insulin-like growth factor 1: 100 ng / mL, B27: 2%, nicotinamide: 30 μM, N-acetylcysteine: 150 μM.

[0015] The special culture medium of the present invention consists of two parts: basic culture medium and culture additives.

[0016] In the basic culture medium, DMEM / F12 culture medium: it is suitable for the growth of various cell types and provides the basic nutrients required by cells; penicillin-streptomycin: used to prevent bacterial contamination during the culture process; HEPES: helps to maintain the pH stability of the culture medium; GlutaMax: as a supplement, it provides amino acids necessary for cell growth.

[0017] Among the culture supplements: epidermal growth factor (EGF): promotes epithelial cell proliferation; basic fibroblast growth factor (bFGF): promotes the proliferation of various cell types; keratinocyte growth factor (KGF): mainly acts on epithelial cells to promote their growth and repair; fibroblast growth factor 10 (FGF10): affects cell proliferation and differentiation; insulin: in addition to regulating glucose uptake, it acts on cell growth and differentiation; transferrin: binds to iron ions to provide the iron required by cells; sodium selenite: provides selenium trace elements to fight oxidative stress; A83-01: used to regulate the cell microenvironment; SB202190: acts on cell behavior; insulin-like growth factor 1 (IGF-1): promotes cell growth and proliferation; B27: contains a variety of growth factors and nutrients; nicotinamide: acts on cell energy metabolism and DNA repair; N-acetylcysteine ​​(NAC): helps protect cells from oxidative stress damage.

[0018] The present invention comprehensively considers various factors such as cell proliferation, differentiation and function maintenance requirements, and develops a culture medium composed of the above components, which is particularly suitable for the long-term culture and research of mammary organoids.

[0019] The present invention also provides a method for preparing the above-mentioned breast cancer organoid-specific culture medium, comprising: first preparing a basal culture medium, then adding culture additives according to the above-mentioned content, and mixing evenly to obtain the culture medium.

[0020] The present invention also provides a method for culturing breast cancer organoids using the above-mentioned breast cancer organoid-specific culture medium, comprising the following steps: (1) Cut the breast cancer tissue sample into small pieces, wash it and then add collagenase for digestion; (2) Resuspend the digested cells in cold Cultrex growth factor reduced BME type 2, add to the cell culture plate, and place in a cell culture incubator to solidify the BME containing the cells; (3) Add the special culture medium to each well, place the cells in a cell culture incubator, replace the special culture medium every 4 to 6 days, and obtain breast cancer organoids in 7 to 14 days.

[0021] Compared with the prior art, the present invention has the following beneficial effects: The present invention is composed of a basic culture medium and a culture additive. Different growth factors are added to the culture additive to simulate the microenvironment of tumor growth in vivo, replacing the activation of the non-specific embryonic stem cell signaling pathway - Wnt / ß-catenin pathway. The special culture medium of the present invention has a better supporting and promoting effect on the growth of breast cancer organoids. Typical breast cancer organoids can be obtained in 7-14 days, and the consistency of patient-derived tumors and tumor heterogeneity are better preserved in vitro, and the stability of the genome of tumor cells cultured in vitro is maintained, laying a solid foundation for further research and application.

[0022] The cost of the special culture medium of the present invention is significantly lower than that of other methods. The relatively expensive recombinant proteins such as the Wnt signal regulating recombinant protein R-Spondin or the developmental regulatory protein Noggin are removed and replaced with other specific components, thereby providing the possibility for subsequent large-scale breast cancer organoid culture for research applications such as drug screening.

[0023] The present invention has been experimentally confirmed that the breast cancer organoids grown in the special culture medium of the present invention are fast, and the diameter grows to 250-300 μm after 7-14 days of growth. The organoid clumps formed grow well and the organoids are uniform in size. At the same time, the present invention uses Ki67 staining, a marker for cell proliferation, to confirm that the cell proliferation rate is about 95%, and uses cleaved caspase-3 staining, a marker for cell apoptosis, to confirm that the cell apoptosis rate is below 5%.

[0024] The present invention can significantly improve the culture success rate of breast cancer organoids, among which: the culture success rate of fine needle aspiration sampling specimens of breast cancer organoids is about 50%, which is more than 25% higher than the existing similar technologies; the culture success rate of surgical specimens of breast cancer organoids is above 90%, which is more than 20% higher than the existing similar technologies.

[0025] In addition, the application method of the special culture medium of the present invention has clear and simple steps, and the operator has little influence on the culture results, which improves the consistency of the culture results and provides convenient conditions for subsequent large-scale culture. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0027] Figure 1 : Representative photos of different fields of view of breast cancer organoids obtained by culturing hollow needle biopsy samples of the right breast of breast cancer patients of the present invention; Figure 2 : Hematoxylin-eosin staining of clinical pathological tissue sections and breast cancer organoids of hollow needle biopsy samples of the right breast of a breast cancer patient of the present invention, c: pathological tissue sections, d: breast cancer organoids; Figure 3 : The breast cancer organoids obtained by in vitro culture of the present invention were long-term subcultured to day 143, and the representative photos of the 13th generation, e: 4x objective lens, f: 20x objective lens; Figure 4 : RNA sequencing results of the present invention. DETAILED DESCRIPTION

[0028] In order to better understand the present invention, the content of the present invention is further clearly described below in conjunction with the embodiments, but the protection content of the present invention is not limited to the following embodiments. In the following description, a large number of specific details are given to provide a more thorough understanding of the present invention. However, it is obvious to those skilled in the art that the present invention can be implemented without one or more of these details. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work premise belong to the scope of protection of the present invention.

[0029] In all examples shown and discussed herein, any specific values ​​should be interpreted as merely exemplary and not as limiting. Therefore, other examples of the exemplary embodiments may have different values.

[0030] Unless otherwise specified, all raw materials are from commercially available products and do not contain other unspecified components except inevitable impurities unless otherwise specified.

[0031] The test method involved in the following case of the present invention is as follows: 1. Fluorescence staining and quantification of Ki67 and cleaved caspase-3: When breast cancer organoids grow to a diameter of 250-300 microns, fix the organoids with 4% paraformaldehyde, then add primary antibodies for Ki67 (host is rabbit) and cleaved caspase-3 (host is mouse), then add secondary antibodies for rabbits conjugated to green fluorescent protein, secondary antibodies for mice conjugated to red fluorescent protein, and nuclear dye 4',6-diamidino-2-phenylindole (DAPI), randomly take photos of 20 fields of view under a fluorescence microscope, calculate the ratio of green (Ki67) to DAPI and the ratio of red fluorescent protein (cleaved caspase-3) to DAPI, and express the cell proliferation rate and cell apoptosis rate in percentage. These two indicators reflect the conditions of cell proliferation (Ki67) and cell apoptosis (cleaved caspase-3), respectively.

[0032] 2. Hematoxylin-eosin staining: When breast cancer organoids grow to a diameter of 250-300 μm, fix them with 4% paraformaldehyde, embed them in conventional paraffin, slice them (4 μm thick), and then stain them sequentially with hematoxylin and eosin.

[0033] 3. RNA sequencing: When breast cancer organoids grow to a diameter of 250-300 microns, digest them with TrypLE Express enzyme, retain the cell clumps after centrifugation, extract RNA with Thermo Fisher's RNA extraction kit, and send them to a sequencing company for whole genome and transcriptome sequencing. After sequencing, perform bioinformatics analysis. Use TrypLE Express enzyme to digest the obtained breast cancer organoids, then passage them at a ratio of 1:4, and then passage them every 2-4 weeks. After long-term culture and passage, perform RNA sequencing.

[0034] The "%" in the following Examples 1-5 represents the volume percentage of the component in the culture medium.

[0035] Example 1: A breast cancer organoid culture medium, comprising a basal culture medium and a culture additive, wherein the basal culture medium is a DMEM / F12 culture medium containing 1% penicillin-streptomycin, 1% HEPES buffer, and 1% GlutaMax solution, and the culture additive comprises the following: epidermal growth factor, basic fibroblast growth factor, keratinocyte growth factor, fibroblast growth factor 10, insulin, transferrin, sodium selenite, A83-01, SB202190, insulin-like growth factor 1, B27, nicotinamide, and N-acetylcysteine, and the culture additive comprises: The mixture was composed of the following components at working concentrations: epidermal growth factor: 5 μg / mL, basic fibroblast growth factor: 10 μg / mL, keratinocyte growth factor: 5 μg / mL, fibroblast growth factor 10: 20 μg / mL, insulin: 10 mg / mL, transferrin: 550 mg / L, sodium selenite: 0.67 mg / L, A83-01: 10 mM, SB202190: 10 mM, insulin-like growth factor 1: 100 ng / mL, B27: 2%, nicotinamide: 30 μM, N-acetylcysteine: 150 μM.

[0036] Example 2: A breast cancer organoid culture medium, comprising a basal culture medium and a culture additive, wherein the basal culture medium is a DMEM / F12 culture medium containing 1% penicillin-streptomycin, 1% HEPES buffer, and 1% GlutaMax solution, and the culture additive comprises the following: epidermal growth factor, basic fibroblast growth factor, keratinocyte growth factor, fibroblast growth factor 10, insulin, transferrin, sodium selenite, A83-01, SB202190, insulin-like growth factor 1, B27, nicotinamide, and N-acetylcysteine; and the culture additive comprises: The nutritional supplement consists of the following ingredients at working concentrations: epidermal growth factor: 1 μg / mL, basic fibroblast growth factor: 20 μg / mL, keratinocyte growth factor: 6 μg / mL, fibroblast growth factor 10: 10 μg / mL, insulin: 20 mg / mL, transferrin: 250 mg / L, sodium selenite: 0.5 mg / L, A83-01: 1 mM, SB202190: 1 mM, insulin-like growth factor 1: 50 ng / mL, B27: 2%, nicotinamide: 5 μM, N-acetylcysteine: 100 μM.

[0037] Example 3: A breast cancer organoid culture medium, comprising a basal culture medium and a culture additive, wherein the basal culture medium is a DMEM / F12 culture medium containing 1% penicillin-streptomycin, 1% HEPES buffer, and 1% GlutaMax solution, and the culture additive comprises the following: epidermal growth factor, basic fibroblast growth factor, keratinocyte growth factor, fibroblast growth factor 10, insulin, transferrin, sodium selenite, A83-01, SB202190, insulin-like growth factor 1, B27, nicotinamide, and N-acetylcysteine, and the culture The supplement consisted of the following components at working concentrations: epidermal growth factor: 3 μg / mL, basic fibroblast growth factor: 30 μg / mL, keratinocyte growth factor: 7 μg / mL, fibroblast growth factor 10: 30 μg / mL, insulin: 30 mg / mL, transferrin: 400 mg / L, sodium selenite: 0.8 mg / L, A83-01: 3 mM, SB202190: 3 mM, insulin-like growth factor 1: 60 ng / mL, B27: 2%, nicotinamide: 15 μM, N-acetylcysteine: 120 μM.

[0038] Example 4: A breast cancer organoid culture medium, comprising a basal culture medium and a culture additive, wherein the basal culture medium is a DMEM / F12 culture medium containing 1% penicillin-streptomycin, 1% HEPES buffer, and 1% GlutaMax solution, and the culture additive comprises the following: epidermal growth factor, basic fibroblast growth factor, keratinocyte growth factor, fibroblast growth factor 10, insulin, transferrin, sodium selenite, A83-01, SB202190, insulin-like growth factor 1, B27, nicotinamide, and N-acetylcysteine, and the culture The supplement consisted of the following components at working concentrations: epidermal growth factor: 8 μg / mL, basic fibroblast growth factor: 40 μg / mL, keratinocyte growth factor: 8 μg / mL, fibroblast growth factor 10: 40 μg / mL, insulin: 40 mg / mL, transferrin: 650 mg / L, sodium selenite: 0.9 mg / L, A83-01: 5 mM, SB202190: 5 mM, insulin-like growth factor 1: 75 ng / mL, B27: 2%, nicotinamide: 40 μM, N-acetylcysteine: 180 μM.

[0039] Example 5: A special culture medium for breast cancer organoids, comprising a basal culture medium and a culture additive, wherein the basal culture medium is a DMEM / F12 culture medium containing 1% penicillin-streptomycin, 1% HEPES buffer, and 1% GlutaMax solution, and the culture additives are composed of the following: epidermal growth factor, basic fibroblast growth factor, keratinocyte growth factor, fibroblast growth factor 10, insulin, transferrin, sodium selenite, A83-01, SB202190, insulin-like growth factor 1, B27, nicotinamide, and N-acetylcysteine, and the culture additives are: The additive consisted of the following components at working concentrations: epidermal growth factor: 10 μg / mL, basic fibroblast growth factor: 50 μg / mL, keratinocyte growth factor: 10 μg / mL, fibroblast growth factor 10: 50 μg / mL, insulin: 50 mg / mL, transferrin: 750 mg / L, sodium selenite: 1.0 mg / L, A83-01: 8 mM, SB202190: 8 mM, insulin-like growth factor 1: 90 ng / mL, B27: 2%, nicotinamide: 50 μM, N-acetylcysteine: 200 μM.

[0040] The method for preparing the breast cancer organoid-specific culture medium provided in Examples 1-5 comprises: first preparing a basal culture medium, then adding culture additives according to the above content, and mixing evenly to obtain the culture medium.

[0041] Example 6: A method for culturing breast cancer organoids in vitro using the dedicated culture medium obtained in Example 1, comprising the following steps: (1) Cut the patient's fine needle aspiration breast cancer tissue sample into 0.5 mm 3 , after washing, collagenase was added for digestion for 60 min; (2) The digested cells were plated at 2×10 6 Resuspend in cold Cultrex growthfactor reduced BME type 2 at a concentration of 10 cells / mL, add 40 μL to each well of a 24-well cell culture plate, and place in a 37°C cell culture incubator for 20 minutes to solidify the BME containing the cells; (3) Add 400 μL of special culture medium to each well and culture in a cell culture incubator with a 5% CO2 concentration and 37°C. Replace the special culture medium every 4 days. Breast cancer organoids can be obtained in 14 days.

[0042] like Figure 1 As shown, representative photographs of different fields of view of the organoids of hollow needle biopsy samples of the right breast of breast cancer patients cultured in Example 6 of the present invention ( Figure 1a, 1b), the breast cancer organoid clumps formed grew well and were uniform in size; after 14 days of culture, the diameter of the breast cancer organoids grew to 250-300μm; pathological results: invasive cancer; immunohistochemistry results: estrogen receptor (strong, +60%), progesterone receptor (weak-medium, +60%), HER2 (-), Ki67 (+10%), P53 (-).

[0043] like Figure 2 As shown, in Example 6 of the present invention, breast cancer organoids cultured from hollow needle biopsy samples of the right breast of breast cancer patients and hematoxylin-eosin staining of patient tissue sections, Figure 2 c is the patient's clinical pathological section, Figure 2 d is an optical microscope morphological image of breast cancer organoids obtained after 14 days of culture. By comparison, the breast cancer organoids obtained after culture are morphologically consistent with the patient's clinical tissue sections.

[0044] The success rate of using the culture medium of this embodiment to culture fine needle aspiration specimens in vitro is about 50%.

[0045] Example 7: A method for culturing breast cancer organoids in vitro using the dedicated culture medium obtained in Example 1, comprising the following steps: (1) Cut the breast cancer tissue sample removed from the patient into 3mm pieces 3 , after washing, collagenase was added for digestion for 60 min; (2) The digested cells were plated at 2×10 6 Resuspend in cold Cultrex growthfactor reduced BME type 2 at a concentration of 10 cells / mL, add 40 μL to each well of a 24-well cell culture plate, and place in a 37°C cell culture incubator for 20 minutes to solidify the BME containing the cells; (3) Add 400 μL of special culture medium to each well and culture in a cell culture incubator with a 5% CO2 concentration and 37°C. Replace the special culture medium every 4 days. Breast cancer organoids can be obtained in 14 days.

[0046] like Figure 3 As shown, a representative photo of the 13th generation of breast cancer organoids obtained by in vitro culture in an embodiment of the present invention after long-term subculture to day 143; Figure 3 e is a 4x objective lens, Figure 3 f is a 20x objective lens.

[0047] like Figure 4 As shown, the RNA sequencing results show the comparison of oncogenic driver genes between tumor organoids after long-term culture and patient tumor tissues. The left side is patient tissue and the right side is tumor organoids, and the sequences of the two are basically the same.

[0048] The success rate of culturing breast cancer organoid surgical specimens in vitro using the culture medium of this example is over 90%.

[0049] The above results indicate that the special culture medium of the present invention has a better supporting and promoting effect on the growth of breast cancer organoids. Typical breast cancer organoids can be obtained in 7-14 days. The consistency of patient-derived tumors and tumor heterogeneity are better preserved in vitro, and the stability of the genome of tumor cells cultured in vitro is maintained, laying the foundation for further research and application.

[0050] The following are comparative examples.

[0051] Comparative Example 1: Compared with Example 1, nicotinamide and N-acetylcysteine ​​were omitted, and other ingredients and their contents remained unchanged.

[0052] The culture medium shown in Comparative Example 1 was used to culture breast cancer organoids in vitro according to the method of Example 6. After 14 days of culture, the diameter of the breast cancer organoids grew to 200-250 μm.

[0053] Comparative Example 2: Compared with Example 1, transferrin and sodium selenite were omitted, and other ingredients and their contents remained unchanged.

[0054] The culture medium shown in Comparative Example 2 was used to culture breast cancer organoids in vitro according to the method of Example 6. After 14 days of culture, the diameter of the breast cancer organoids grew to 200-250 μm.

[0055] Comparative Example 3: Compared with Example 1, insulin-like growth factor 1 was omitted, and BPE was selected to replace insulin-like growth factor 1, while other components and contents remained unchanged.

[0056] The culture medium shown in Comparative Example 3 was used to culture breast cancer organoids in vitro according to the method of Example 6. After 14 days of culture, the diameter of the breast cancer organoids grew to 200-250 μm.

[0057] Comparative Example 4: Compared with Example 1, nicotinamide, N-acetylcysteine, transferrin and sodium selenite were omitted, and other ingredients and their contents remained unchanged.

[0058] The culture medium shown in Comparative Example 4 was used to culture breast cancer organoids in vitro according to the method of Example 6. After 14 days of culture, the diameter of the breast cancer organoids grew to 150-200 μm.

[0059] Comparative Example 5: Referring to CN 113583961 A, a serum-free culture medium for breast cancer organoids without the need to add Wnt pathway regulating recombinant proteins and Noggin, the culture medium includes a serum-free basal culture medium and a culture additive. The basal culture medium is a DMEM / F12 culture medium containing 1% penicillin-streptomycin, 1% HEPES buffer, and 1% GlutaMax solution, and the culture additive is composed of the following working concentrations: EGF: 50μg / mL, FGF2: 10μg / mL, FGF7: 5μg / mL, FGF10: 20μg / mL, Neuregulin1: 5μM, insulin: 10mg / mL, hydrocortisone: 1mg / mL, small molecule inhibitor A83-01: 10mM, Y-27632: 10mM, SB202190: 10mM, BPE: 1%, B27: 2%, ITS: 1%.

[0060] The culture medium shown in Comparative Example 5 was used to culture breast cancer organoids in vitro according to the method of Example 6. After 14 days of culture, the diameter of the breast cancer organoids grew to 150-200 μm. The success rate of surgical specimens of breast cancer organoids cultured in vitro using the culture medium of Comparative Example 5 was about 70%; the success rate of fine needle aspiration specimens was about 25%, which shows that the success rate of surgical specimens of breast cancer organoids of the present invention is more than 20% higher than that of the prior art, and the success rate of fine needle aspiration specimens is about 25% higher than that of the prior art.

[0061] The cell proliferation rate and cell apoptosis rate results of the above Example 6 and Comparative Examples 1-5 are shown in the following table: The test results in the table show that the cell proliferation rate of the present invention is 95.3% confirmed by staining with Ki67, a cell proliferation marker, and the cell apoptosis rate is below 5% confirmed by staining with cleaved caspase-3, a cell apoptosis marker. The above results are significantly different from those of Comparative Examples 1-5, respectively, indicating that the culture medium of the present invention provides an environment conducive to the growth and survival of breast cancer cells, thereby enabling the cells to maintain a high proliferation rate and a low apoptosis rate during the culture process.

[0062] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Other modifications or equivalent substitutions made to the technical solution of the present invention by ordinary technicians in the field should be included in the scope of the claims of the present invention as long as they do not depart from the spirit and scope of the technical solution of the present invention.

Claims

1. A breast cancer organoid culture medium, comprising a basal culture medium and a culture additive, wherein the basal culture medium is a DMEM / F12 culture medium, characterized in that: The culture additive consists of epidermal growth factor, basic fibroblast growth factor, keratinocyte growth factor, fibroblast growth factor 10, insulin, transferrin, sodium selenite, A83-01, SB202190, insulin-like growth factor 1, B27, nicotinamide and N-acetylcysteine.

2. A breast cancer organoid culture medium according to claim 1, characterized in that: The DMEM / F12 culture medium contains 1% penicillin-streptomycin, 1% HEPES buffer and 1% GlutaMax solution.

3. A breast cancer organoid-specific culture medium according to claim 2, characterized in that: The working concentration of the epidermal growth factor is 1-10 μg / mL, the working concentration of the basic fibroblast growth factor is 10-50 μg / mL, the working concentration of the keratinocyte growth factor is 5-10 μg / mL, the working concentration of the fibroblast growth factor 10 is 10-50 μg / mL, the working concentration of the insulin is 10-50 mg / mL, the working concentration of the transferrin is 250-750 mg / L, the working concentration of the sodium selenite is 0.5-1.0 mg / L, the working concentration of the A83-01 is 1-10 mM, the working concentration of the SB202190 is 1-10 mM, the working concentration of the insulin-like growth factor 1 is 50-100 ng / mL, the working concentration of the B27 is 2%, the working concentration of the nicotinamide is 5-50 μM, and the working concentration of the N-acetylcysteine ​​is 100-200 μM.

4. A breast cancer organoid-specific culture medium according to claim 3, characterized in that: The culture supplement consists of the following ingredients at working concentrations: Composition: Epidermal growth factor: 5μg / mL, basic fibroblast growth factor: 10μg / mL, keratinocyte growth factor: 5μg / mL, fibroblast growth factor 10: 20μg / mL, insulin: 10mg / mL, transferrin: 550mg / L, sodium selenite: 0.67mg / L, A83-01: 10mM, SB202190: 10mM, insulin-like growth factor 1: 100ng / mL, B27: 2%, nicotinamide: 30μM, N-acetylcysteine: 150μM.

5. A method for culturing breast cancer organoids using a breast cancer organoid-specific culture medium according to any one of claims 1 to 4, characterized in that: The following steps are involved: Breast cancer tissue samples were cut into small pieces, washed, and digested with collagenase; Resuspend the digested cells in cold Cultrex growth factor reduced BME type 2, add to the cell culture plate, and place in the cell culture incubator to solidify the BME containing the cells; The special culture medium is added to each well, and the cells are placed in a cell culture incubator for culture. The special culture medium is replaced every 4-6 days, and breast cancer organoids are obtained after culture for 7-14 days.

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