A special culture medium for breast cancer organoids and its culture method

Abstract

The present invention provides a special culture medium for breast cancer organoids and a culture method thereof, belonging to the technical field of primary cell culture. A special culture medium for breast cancer organoids is composed of a basal medium and culture additives. The basal medium is DMEM / F12 medium, and the culture additives are composed 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. The culture medium of the present invention improves the stability of the tumor cell genome during the culture while reducing the culture cost.

Description

Technical Field

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

[0002] With the extension of human lifespan and the change of lifestyle, the incidence and fatality rate of cancer have been increasing year by year. Among women, the incidence of breast cancer is the highest and shows an increasing trend, seriously threatening women's health. Breast cancer is a tumor derived from breast epithelial cells. Early on, pathologists recognized that breast cancer has a high degree of tumor heterogeneity. One manifestation of tumor heterogeneity is the large difference between different tumors. Based on this, breast cancer is divided into different types to guide clinical medication and prognosis judgment.

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

[0004] In the past few decades, researchers have developed different culture methods to strive to most accurately reflect the in vivo status of tumors. The culture of organoids is a culture method in which cells or tissues are cultured in vitro to form a 3D structure and form a structure similar to the cell source or tissue source. The earliest was the culture of colon cells. Normal colon stem cells can be cultured and differentiated into colon villus-like structures in vitro and maintain genomic stability. Soon this technology was used to culture colon cancer cells and 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 in vitro tumor model can accurately simulate the biological characteristics of in vivo tumors and the response to drug treatment, and has great value for tumor research, drug testing and screening.

[0006] Although great progress has been made in the current cultivation of tumor organoids, there are still limitations in its cultivation methods. The cultivation principle of tumor organoids is usually to activate the Wnt / β-catenin pathway of cells, so as to enable cell proliferation 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 genomic stability of tumor cells, thereby changing the mutation spectrum of tumors. It has been confirmed by research scholars that after long-term subculture, the gene mutation spectrum of tumors has drifted compared with the original tumors. This gene mutation drift may be related to various factors, including culture conditions, selective growth of cells, and changes in the cell microenvironment, etc.

[0007] In addition, it is generally necessary to add recombinant proteins such as R-Spondin, a regulator of Wnt signaling, or Noggin, a developmental regulatory protein, to the main components of breast cancer organoid culture media. For example, a breast cancer organoid culture kit disclosed in patent document CN 111500540A contains Noggin and one or more of the secreted protein family R-Spondin1-4 related to the Wnt signaling pathway. Such recombinant proteins are expensive, and the main production enterprises are all located abroad, which leads to problems such as high production costs, the need to import the main added components, long procurement cycles, and the supply cycle being easily affected by the external environment for breast cancer organoid-specific culture media. These problems have greatly hindered the large-scale cultivation and wider application research of breast cancer organoids.

[0008] Furthermore, there are also problems such as low cultivation success rate and low passage efficiency in breast cancer organoid culture. The cultivation success rate of breast cancer organoids reported in existing research (Drost J, Clevers H. Organoids in cancer research[J]. Nat Rev Cancer, 2018,18(7): 407-418.) is about 60%. The cultivation success rate of breast cancer organoids reported by 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.) can reach 80%, but its cultivation and passage methods are cumbersome and the cultivation cost is high, which limits its popularization and application.

[0009] Therefore, there is an urgent need to develop breast cancer organoids with lower costs and easier usability, as well as a dedicated culture medium that can better maintain the tumor gene mutation map. 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. Therefore, the development of a culture medium for normal breast organoids is also imminent. Summary of the Invention

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

[0011] To solve the above technical problems, the technical solution adopted by the present invention is:

[0012] A dedicated culture medium for breast cancer organoids, which is composed of a basal medium and culture additives. The basal medium is DMEM / F12 medium, and the culture additives are composed 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.

[0013] Optionally, the DMEM / F12 medium contains 1% penicillin-streptomycin, 1% HEPES buffer, and 1% GlutaMax solution.

[0014] 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;

[0015] The working concentration of the basic fibroblast growth factor is 10-50 μg / mL, optionally 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;

[0016] The working concentration of the keratinocyte growth factor is 5-10 μg / mL, optionally 5 μg / mL, 6 μg / mL, 7 μg / mL, 8 μg / mL, 9 μg / mL, or 10 μg / mL, preferably 5-8 μg / mL;

[0017] The working concentration of the fibroblast growth factor 10 is 10 - 50 μg / mL, and it 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;

[0018] The working concentration of the insulin is 10 - 50 mg / mL, and it 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;

[0019] The working concentration of the transferrin is 250 - 750 mg / L, and it 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;

[0020] The working concentration of the sodium selenite is 0.5 - 1.0 mg / L, and it 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;

[0021] The working concentration of the A83 - 01 is 1 - 10 mM, and it can be 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;

[0022] The working concentration of the SB202190 is 1 - 10 mM, and it can be 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;

[0023] The working concentration of the insulin - like growth factor 1 is 50 - 100 ng / mL, and it can be 50 ng / mL, 60 ng / mL, 70 ng / mL, 80 ng / mL, 90 ng / mL or 100 ng / mL, preferably 75 - 100 ng / mL;

[0024] The working concentration of the B27 is 2%;

[0025] The working concentration of the nicotinamide is 5 - 50 μM, and it 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;

[0026] The working concentration of the N-acetylcysteine is 100-200 μM, and it can be optionally 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.

[0027] Preferably, the culture additive consists of the following components at their 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%, niacinamide: 30 μM, N-acetylcysteine: 150 μM.

[0028] The special medium of the present invention consists of two parts: a basal medium and a culture additive.

[0029] In the basal medium, DMEM / F12 medium: it is suitable for the growth of various cell types and provides the basic nutrients required by the cells; penicillin-streptomycin: used to prevent bacterial contamination during the culture process; HEPES: helps to maintain the pH stability of the culture solution; GlutaMax: as a supplement, provides the essential amino acids for cell growth.

[0030] In the culture additive: epidermal growth factor (EGF): promotes the proliferation of epithelial cells; 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, acts on cell growth and differentiation; transferrin: binds to iron ions and provides the iron required by the cells; sodium selenite: provides the trace element selenium and counteracts 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 various growth factors and nutrients; niacinamide: acts on cell energy metabolism and DNA repair; N-acetylcysteine (NAC): helps to protect cells from oxidative stress damage.

[0031] The present invention comprehensively considers various factors such as cell proliferation, differentiation and function maintenance requirements, and develops a medium composed of the above components, which is particularly suitable for the long-term culture and research of mammary organoids.

[0032] The present invention also provides a method for preparing the above-mentioned special culture medium for breast cancer organoids, which includes: first preparing a basal medium, and then adding culture additives according to the above contents and mixing evenly to obtain the medium.

[0033] The present invention also provides a method for culturing breast cancer organoids by using the above-mentioned special culture medium for breast cancer organoids, which includes the following steps:

[0034] (1) Cutting a breast cancer tissue sample into small pieces, washing and then adding collagenase for digestion;

[0035] (2) Resuspending the digested cells in cold Cultrex growth factor reduced BME type2, adding them to a cell culture plate, and placing them in a cell culture incubator to solidify the BME containing the cells;

[0036] (3) Adding the special culture medium to each well, culturing in a cell culture incubator, replacing the special culture medium every 4-6 days, and obtaining breast cancer organoids in 7-14 days.

[0037] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0038] The present invention is composed of a basal medium and culture additives. In the culture additives, different growth factors are added 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, which better retains the consistency of patient-derived tumors and tumor heterogeneity in vitro, maintains the stability of the genome of tumor cells in vitro culture, and lays a solid foundation for further research and application.

[0039] The cost of the special culture medium of the present invention is significantly reduced compared with other methods. Expensive recombinant proteins such as Wnt signal regulatory recombinant protein R-Spondin or developmental regulatory protein Noggin are removed, and other specific components are used for replacement, thus providing the possibility for subsequent large-scale culture of breast cancer organoids for research applications such as drug screening.

[0040] The present invention has been experimentally verified that when the special culture medium of the present invention is used for culturing breast cancer organoids, the growth rate of breast cancer organoids is fast. After growing for 7 - 14 days, the diameter grows to 250 - 300 μm. The formed organoid aggregates grow well and the sizes of the organoids are uniform. At the same time, the present invention uses Ki67 staining of cell proliferation markers to confirm that the cell proliferation rate is about 95%, and uses cleaved caspase-3 staining of apoptosis to confirm that the apoptosis rate is below 5%.

[0041] The present invention can significantly improve the success rate of culturing breast cancer organoids. Among them, the success rate of culturing fine needle aspiration biopsy specimens of breast cancer organoids is about 50%, which is more than 25% higher than the existing similar technologies; the success rate of culturing surgical specimens of breast cancer organoids is more than 90%, which is more than 20% higher than the existing similar technologies.

[0042] In addition, the application method steps of the special culture medium of the present invention are clear and simple, and the influence of operators on the culture results is small, improving the consistency of the culture results and providing convenient conditions for subsequent large-scale culture. Description of the Drawings

[0043] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0044] Figure 1 : Representative photos of different fields of view of breast cancer organoids obtained by culturing the right breast core needle biopsy samples of breast cancer patients of the present invention;

[0045] Figure 2 : Hematoxylin-eosin staining diagrams of the clinical pathological tissue sections and breast cancer organoids of the right breast core needle biopsy samples of breast cancer patients of the present invention, c: pathological tissue section, d: breast cancer organoid;

[0046] Figure 3 : Representative photos of the long-term subculture of breast cancer organoids obtained by in vitro culture of the present invention to the 143rd day, 13th generation, e: 4× objective lens, f: 20× objective lens;

[0047] Figure 4 : RNA sequencing results of the present invention. Detailed Embodiments

[0048] To better understand the present invention, the content of the present invention will be further clearly elaborated below in conjunction with embodiments. However, the protected 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. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts fall within the scope of protection of the present invention.

[0049] In all the examples shown and discussed here, any specific value should be construed as merely exemplary, not as a limitation. Therefore, other examples of the exemplary embodiments may have different values.

[0050] Unless otherwise specified, all raw materials are commercially available products, and unless otherwise specified, they do not contain other components not explicitly mentioned except for inevitable impurities.

[0051] The test methods involved in the following cases of the present invention are as follows:

[0052] 1. Fluorescent staining and quantification of Ki67 and cleaved caspase-3: When the breast cancer organoids grow to a diameter of 250 - 300 microns, the organoids are fixed with 4% paraformaldehyde, and then the primary antibodies of Ki67 (host: rabbit) and cleaved caspase-3 (host: mouse) are added. Then, the secondary antibody of rabbit conjugated with green fluorescent protein, the secondary antibody of mouse conjugated with red fluorescent protein, and the nuclear dye 4',6-diamidino-2-phenylindole (DAPI) are added. Randomly take 20 fields of view under a fluorescence microscope, and calculate the ratio of green (Ki67) to DAPI and the ratio of red fluorescent protein (cleaved caspase-3) to DAPI, which represent the cell proliferation rate and cell apoptosis rate in percentage. These two indicators respectively reflect the situations of cell proliferation (Ki67) and cell apoptosis (cleaved caspase-3).

[0053] 2. Hematoxylin-eosin staining: When the breast cancer organoids grow to a diameter of 250 - 300 microns, the organoids are fixed with 4% paraformaldehyde, routinely paraffin-embedded, sectioned (with a thickness of 4 microns), and then sequentially placed in hematoxylin and eosin for staining.

[0054] 3. RNA sequencing: When the breast cancer organoids grow to a diameter of 250 - 300 microns, digest the breast cancer organoids with TrypLE Express enzyme, centrifuge and retain the cell clumps, extract RNA using the RNA extraction kit from Thermo Fisher Scientific, send it to a sequencing company for whole genome and transcriptome sequencing, and perform bioinformatics analysis after sequencing. Digest the obtained breast cancer organoids with TrypLE Express enzyme, 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.

[0055] In the following Examples 1 - 5, “%” represents the volume percentage of the component in the culture medium.

[0056] Example 1: A special culture medium for breast cancer organoids, which consists of a basal medium and culture additives. The basal medium is DMEM / F12 medium containing 1% penicillin - streptomycin, 1% HEPES buffer, and 1% GlutaMax solution. 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. The culture additives are composed of the following components at the 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.

[0057] Example 2: A special medium for breast cancer organoids, which is composed of a basal medium and culture additives. The basal medium is DMEM / F12 medium containing 1% penicillin-streptomycin, 1% HEPES buffer, and 1% GlutaMax solution. The culture additives are composed 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. The culture additives are composed of the following components 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.

[0058] Example 3: A special medium for breast cancer organoids, which is composed of a basal medium and culture additives. The basal medium is DMEM / F12 medium containing 1% penicillin-streptomycin, 1% HEPES buffer, and 1% GlutaMax solution. The culture additives are composed 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. The culture additives are composed 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.

[0059] Example 4: A special medium for breast cancer organoids, which is composed of a basal medium and culture additives. The basal medium is DMEM / F12 medium containing 1% penicillin-streptomycin, 1% HEPES buffer, and 1% GlutaMax solution. 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. The culture additives are composed of the following components at the 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.

[0060] Example 5: A special medium for breast cancer organoids, which is composed of a basal medium and culture additives. The basal medium is DMEM / F12 medium containing 1% penicillin-streptomycin, 1% HEPES buffer, and 1% GlutaMax solution. 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. The culture additives are composed of the following components at the 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.

[0061] The preparation method of the special medium for breast cancer organoids provided in Examples 1-5 includes: first preparing the basal medium, and then adding the culture additives according to the above contents and mixing evenly to obtain the medium.

[0062] Example 6: A method for culturing breast cancer organoids in vitro using the special medium obtained in Example 1 above, which includes the following steps:

[0063] (1) Cut the breast cancer tissue sample obtained by fine needle aspiration of the patient into 0.5 mm 3 , and after washing, add collagenase and digest for 60 min;

[0064] (2) Resuspend the digested cells at a concentration of 2×10 6 cells / mL in cold Cultrex growth factor reduced BME type 2, add to a 24-well cell culture plate, add 40 μL to each well, and place in a 37 °C cell culture incubator for 20 minutes to solidify the BME containing the cells;

[0065] (3) Add 400 μL of the special medium to each well, place in a 5% CO2 concentration and 37 °C cell culture incubator for culture, change the special medium every 4 days, and breast cancer organoids can be obtained in 14 days.

[0066] As Figure 1 shown, representative photos of different fields of view of the organoids of the right breast core needle biopsy sample of breast cancer patients cultured in Example 6 of the present invention ( Figure 1 a, 1b), the formed breast cancer organoid masses grow well and are uniform in size; after culturing for 14 days, the diameter of the breast cancer organoids grows to 250-300 μm; pathological result: invasive carcinoma; immunohistochemical result: estrogen receptor (strong, +60%), progesterone receptor (weak-medium, +60%), HER2 (-), Ki67 (+10%), P53 (-).

[0067] As Figure 2 shown, the breast cancer organoids of the right breast core needle biopsy sample of breast cancer patients cultured in Example 6 of the present invention and the patient tissue sections were stained with hematoxylin-eosin, Figure 2 c is the patient's clinical pathological section, Figure 2 d is the optical microscope morphology diagram of the breast cancer organoids obtained after culturing for 14 days. By comparison, it can be seen that the breast cancer organoids obtained by culturing are consistent with the patient's clinical tissue sections in terms of morphology.

[0068] The success rate of culturing the fine needle aspiration specimens in vitro using the medium of this example is about 50%.

[0069] Example 7: A method for culturing breast cancer organoids in vitro using the special medium obtained in Example 1 above, including the following steps:

[0070] (1) Cut the breast cancer tissue sample obtained by surgical resection of the patient into 3 mm 3 , and after washing, add collagenase and digest for 60 min;

[0071] (2) Resuspend the digested cells at a concentration of 2×10 6Resuspend at a concentration of cells / mL in cold Cultrex growth factor reduced BME type 2, add to a 24-well cell culture plate, add 40 μL to each well, and place in a 37 °C cell culture incubator for 20 minutes to solidify the BME containing the cells;

[0072] (3) Add 400 μL of the special medium to each well, place in a cell culture incubator with a 5% CO2 concentration at 37 °C for culturing, change the special medium every 4 days, and breast cancer organoids can be obtained in 14 days.

[0073] As Figure 3 shown, representative photos of the 13th generation of breast cancer organoids obtained by in vitro culture in the examples of the present invention after long-term subculture to the 143rd day; Figure 3 e is a 4× objective lens, Figure 3 f is a 20× objective lens.

[0074] As Figure 4 shown, the RNA sequencing results show the comparison of the oncogenic driver genes between the tumor organoids after long-term subculture and the patient's tumor tissue. The left side is the patient's tissue, and the right side is the tumor organoids. The sequences of the two are basically the same.

[0075] The success rate of in vitro culturing breast cancer organoid surgical specimens using the medium of this example is above 90%.

[0076] The above results show that the special 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, better retaining the tumor consistency and tumor heterogeneity from the patient in vitro, maintaining the stability of the genome of tumor cells in vitro culture, and laying a foundation for further research and application.

[0077] The following are comparative examples.

[0078] Comparative Example 1: Compared with Example 1, nicotinamide and N-acetylcysteine are omitted, and other components and contents remain unchanged.

[0079] Using the medium shown in Comparative Example 1, in vitro culture of breast cancer organoids was carried out according to the method of Example 6. When cultured for 14 days, the diameter of the breast cancer organoids grew to 200 - 250 μm.

[0080] Comparative Example 2: Compared with Example 1, transferrin and sodium selenite are omitted, and other components and contents remain unchanged.

[0081] Using the medium shown in Comparative Example 2, in vitro culture of breast cancer organoids was carried out according to the method of Example 6. When cultured for 14 days, the diameter of the breast cancer organoids grew to 200 - 250 μm.

[0082] 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 their contents remained unchanged.

[0083] Using the medium shown in Comparative Example 3, the in vitro culture of breast cancer organoids was carried out according to the method of Example 6. When cultured for 14 days, the diameter of the breast cancer organoids grew to 200 - 250 μm.

[0084] Comparative Example 4: Compared with Example 1, niacinamide, N-acetylcysteine, transferrin, and sodium selenite were omitted, while other components and their contents remained unchanged.

[0085] Using the medium shown in Comparative Example 4, the in vitro culture of breast cancer organoids was carried out according to the method of Example 6. When cultured for 14 days, the diameter of the breast cancer organoids grew to 150 - 200 μm.

[0086] Comparative Example 5: Referring to CN 113583961 A, a serum-free special medium for breast cancer organoids without adding Wnt pathway regulatory recombinant protein and noggin. The medium includes a serum-free basal medium and culture additives. The basal medium is DMEM / F12 medium containing 1% penicillin-streptomycin, 1% HEPES buffer, and 1% GlutaMax solution. The culture additives are composed of the following components at working concentrations: EGF: 50 μg / mL, FGF2: 10 μg / mL, FGF7: 5 μg / mL, FGF10: 20 μg / mL, Neuregulin1: 5 μM, insulin: 10 mg / mL, hydrocortisone: 1 mg / mL, small molecule inhibitor A83-01: 10 mM, Y-27632: 10 mM, SB202190: 10 mM, BPE: 1%, B27: 2%, ITS: 1%.

[0087] Using the medium shown in Comparative Example 5, the in vitro culture of breast cancer organoids was carried out according to the method of Example 6. When cultured for 14 days, the diameter of the breast cancer organoids grew to 150 - 200 μm. The success rate of in vitro culturing breast cancer organoid surgical specimens using the medium of Comparative Example 5 was about 70%; the success rate of fine needle aspiration sampling specimens was about 25%. This indicates that the success rate of breast cancer organoid surgical specimens of the present invention is increased by more than 20% compared with the prior art, and the success rate of fine needle aspiration sampling specimens is increased by about 25% compared with the prior art.

[0088] The results of the cell proliferation rate and cell apoptosis rate of the above Example 6 and Comparative Examples 1 - 5 are shown in the following table:

[0089]

[0090] The test results in the table show that the cell proliferation rate of 95.3% was confirmed by Ki67 staining, a marker of cell proliferation, in the present invention. At the same time, the apoptosis rate was confirmed to be below 5% by cleaved caspase-3 staining for apoptosis. Compared with Comparative Examples 1-5 respectively, the above results all showed significant differences, indicating that the culture medium of the present invention provided a favorable environment for the growth and survival of breast cancer cells, thus enabling the cells to maintain a high proliferation rate and a low apoptosis rate during the culture process.

[0091] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Any other modifications or equivalent replacements made by those of ordinary skill in the art to the technical solutions of the present invention shall be covered within the scope of the claims of the present invention as long as they do not depart from the spirit and scope of the technical solutions of the present invention.

Claims

1. A special culture medium for breast cancer organoids, which is composed of a basal medium and culture additives, and is characterized in that: The basal medium is DMEM / F12 medium, which contains 1% penicillin-streptomycin, 1% HEPES buffer, and 1% GlutaMax solution, and does not contain the Wnt signaling regulatory recombinant protein R-Spondin or the developmental regulatory protein Noggin. 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. The working concentration of the epidermal growth factor is 1-5 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 insulin is 10-50 mg / mL, the working concentration of transferrin is 250-750 mg / L, the working concentration of sodium selenite is 0.5-1.0 mg / L, the working concentration of A83-01 is 1-10 mM, the working concentration of SB202190 is 1-10 mM, the working concentration of insulin-like growth factor 1 is 50-100 ng / mL, the working concentration of B27 is 2%, the working concentration of nicotinamide is 5-50 μM, and the working concentration of N-acetylcysteine is 100-200 μM.

2. The dedicated culture medium for breast cancer organoids according to claim 1, wherein: The culture additive consists of components at the following working concentrations as follows: 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.

3. A method for culturing breast cancer organoids using a special culture medium for breast cancer organoids as described in claim 1 or 2, characterized in that: It includes the following steps: Cut the breast cancer tissue sample into small pieces, wash it, and add collagenase for digestion; Resuspend the digested cells in cold Cultrex growth factor reduced BME type2, add them to a cell culture plate, and place it in a cell culture incubator to solidify the BME containing the cells; Add the special medium to each well, place it in a cell culture incubator for culture, replace the special medium every 4-6 days, and culture for 7-14 days to obtain breast cancer organoids.

Images