A culture medium, method and application for establishing mammary organoids or / and breast cancer organoids

Abstract

The invention discloses a culture medium, a method and an application for establishing a mammary organoid or / and a breast cancer organoid, and belongs to the technical field of biomedicine. The culture medium is a DMEM / F12 complete culture medium containing the following components: estradiol, hydrocortisone, R-Spondin 3, Neuregulin 1, FGF7, FGF10, rh-EGF, PDGF, Noggin, A83-01, Wnt3a, Y-27632, SB202190, B27, N-acetylcysteine, nicotinamide, GlutaMax, fetal bovine serum and penicillin / streptomycin double antibody. The culture medium is used for establishing a mammary organoid or / and a breast cancer organoid, can accelerate the proliferation and self-assembly ability of mammary or / and breast cancer cells, accelerate the establishment of organoids and improve the success rate of the establishment of organoids.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a culture medium, method and application for establishing mammary organoids or / and breast cancer organoids. Background Art

[0002] Breast cancer is a phenomenon in which mammary epithelial cells proliferate uncontrollably under the action of multiple carcinogens. In the early stages of the disease, symptoms such as breast lumps, nipple discharge, and axillary lymph node enlargement often occur. In the late stages, cancer cells may metastasize to distant sites, resulting in multi-organ lesions that directly threaten the patient's life. Breast cancer is often called the "pink killer" and its incidence ranks first among female malignant tumors. Male breast cancer is relatively rare. At present, the level of anti-breast cancer drugs is uneven. Effective drugs are very expensive and the therapeutic effect is not ideal. The level of drug screening is uneven. There is a lack of an evaluation system that can highly simulate the in vivo microenvironment. Organoid culture is increasingly used to experimentally simulate human cancer in order to customize personalized drugs and predict drug responses. Breast cancer is no exception, but primary breast cancer in particular has some inherent difficulties. Residual non-malignant cells are often present in biopsies. Recently, organoid technology has been used to generate a large number of breast cancer organoids. Compared with 2D cell culture, the three-dimensional culture of organoids can better reflect the growth environment of tumor tissue in the body, and both the structure and subcellular types are highly restored to the original ecology of tumor organs.

[0003] Organoids are differentiated from pluripotent stem cells or organ progenitor cells and self-assembled into complete mammalian organs with structure and function. Organoid technology has great potential in studying a wide range of subjects, including developmental biology, disease pathology, cell biology, regeneration mechanisms, precision medicine, and drug toxicity and efficacy testing. For these and other applications, organoid culture achieves high-information complementarity to existing 2D culture methods and animal model systems, and is an important bridge between two-dimensional culture and in vivo models. It is more physiologically relevant than monolayer cell culture models, and is easier to manipulate than in vivo models to monitor signal pathways and genome editing. The value of organoids lies in their ability to self-organize into the smallest biological unit, showing functions and complexity similar to the original tissue. The operability of organoids indicates that organoids will provide an excellent model system for a wide range of basic research, including expression profiling studies and analysis of rare cell lineages that are difficult to obtain in vivo.

[0004] Organoids have shown great potential in the field of anti-tumor drug screening. Drug screening tests show the most intuitive performance of drugs on organoids, which highly simulates the effects of drugs on organs. Therefore, there is an urgent need for a culture method that can accelerate the proliferation and self-assembly ability of breast cancer cells, accelerate the establishment of organoids, and improve the success rate of organoid establishment, providing diversity for the cultivation of breast cancer organoids. Summary of the invention

[0005] In view of this, one of the objects of the present invention is to provide a culture medium for establishing mammary organoids or / and breast cancer organoids; a second object of the present invention is to provide a method for establishing mammary organoids or / and breast cancer organoids based on the culture medium; a third object of the present invention is to provide the use of the culture medium in breast cancer organoids or / and mammary organoids for screening anti-breast cancer drugs.

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] 1. A culture medium for establishing mammary organoids or / and breast cancer organoids

[0008] The culture medium is a DMEM / F12 complete culture medium containing the following components: estradiol, hydrocortisone, R-Spondin3, Neuregulin 1, FGF7, FGF10, rh-EGF, PDGF, Noggin, A83-01, Wnt3a, Y-27632, SB202190, B27, N-acetylcysteine, nicotinamide, GlutaMax, fetal bovine serum, and penicillin / streptomycin double antibody.

[0009] Preferably, the concentrations of the components are as follows: estradiol 10nM-1000nM, hydrocortisone 0.1-100μg / ml, R-Spondin 3 100-500ng / ml, Neuregulin 1 1-10nM, FGF7 1-10ng / ml, FGF10 10-100ng / ml, rh-EGF1-10ng / ml, PDGF 1-10ng / ml, Noggin 50-500ng / ml, A83-01 100-1000nM, Wnt3a 1-5nM, Y-276321-10μM, SB202190 100-1000ng, B27 10ng / ml, N-acetylcysteine ​​1-5nM, nicotinamide 1-10nM, GlutaMax 1-5nM, fetal bovine serum volume fraction 10%, penicillin / streptomycin double antibody volume fraction 1%.

[0010] 2. A method for establishing mammary organoids or / and breast cancer organoids based on the culture medium, comprising the following steps:

[0011] (1) preparing the obtained breast tissue or breast cancer tissue into a single cell suspension;

[0012] (2) The single cell suspension prepared in step (1) is added to the 3D culture matrix gel and resuspended, then inoculated into a culture device, the culture medium is added, and the culture is expanded in an incubator.

[0013] Preferably, in step (1) of the present invention, the breast tissue or breast cancer tissue is derived from a mammal.

[0014] Preferably, in step (1), the breast tissue or breast cancer tissue is prepared into a single cell suspension by shearing, grinding and filtering.

[0015] It is further preferred that in step (1), the size of the shredded tissue is 1 cm×1 cm.

[0016] It is further preferred that in step (1), the filtration is performed using a 200-mesh filter.

[0017] Preferably, in step (2) of the present invention, the culture medium is replaced every three days during the expanded culture.

[0018] 3. Use of the culture medium in screening anti-breast cancer drugs using breast cancer organoids and / or mammary gland organoids.

[0019] Preferably, the drug of the present invention comprises chemotherapy drugs, radiotherapy drugs and / or targeted drugs.

[0020] The beneficial effects of the present invention are as follows: the present invention discloses a culture medium, method and application for establishing mammary organoids or / and breast cancer organoids, wherein the culture medium is a DMEM / F12 complete culture medium containing the following components: estradiol, hydrocortisone, R-Spondin 3, Neuregulin 1, FGF7, FGF10, rh-EGF, PDGF, Noggin, A83-01, Wnt3a, Y-27632, SB202190, B27, N-acetylcysteine, nicotinamide, GlutaMax, fetal bovine serum and penicillin / streptomycin dual antibody, and the culture medium is used for establishing mammary organoids or / and breast cancer organoids, can accelerate the proliferation and self-assembly ability of mammary or / and breast cancer cells, improve the establishment of organoids and improve the success rate of organoid establishment. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to make the purpose, technical solution and beneficial effects of the present invention clearer, the present invention provides the following drawings for illustration:

[0022] Figure 1 Growth morphology of breast cancer organoids cultured in the medium of formula 1-3 for 14 days (100 times);

[0023] Figure 2 This is a growth curve of breast cancer organoids cultured in the medium of formula 1-3 for 14 days;

[0024] Figure 3The growth morphology of breast cancer organoids cultured in culture medium without the important growth factor PDGF for 14 days for comparison (100 times);

[0025] Figure 4 This is a growth curve of breast cancer organoids cultured in a medium without the important growth factor PDGF for 14 days in comparison with Formula 1;

[0026] Figure 5 The growth morphology of breast cancer organoids cultured in culture medium without important growth factors Wnt3a, FGF 7, FGF 10, PDGF, and rh-EGF for 14 days is compared with Formula 2;

[0027] Figure 6 This is a growth curve of breast cancer organoids cultured for 14 days in a culture medium containing the important growth factors Wnt3a, FGF 7, FGF 10, PDGF, and rh-EGF compared with Formula 2;

[0028] Figure 7 Growth morphology of mammary organoids cultured in the medium of formula 1-3 for 3 days, 5 days, and 10 days (100 times);

[0029] Figure 8 HE staining of breast cancer organoids cultured in the medium of formula 1-3 on the 14th day (400 times);

[0030] Fig. 9 CCK-8 tumor inhibition rate graph for screening anti-tumor drugs for breast cancer organoids; DETAILED DESCRIPTION

[0031] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0032] The culture medium components involved in the embodiments of the present invention include estradiol (β-estradiol), hydrocortisone, R-Spondin 3, and Neuregulin from R&D. 1, purchased from Peprotech; FGF7, purchased from Peprotech; FGF10, purchased from Peprotech; rh-EGF, purchased from Peprotech; PDGF, purchased from Peprotech; Noggin, purchased from Peprotech; A83-01, purchased from Tocris; Wnt3a, purchased from Gibco; Y-27632, purchased from Abmole; SB202190, purchased from Sigma; B27, purchased from Gibco; N-Acetylcysteine, purchased from Gibco; Nicotinamide, purchased from Sigma; GlutaMax, purchased from Sigma; FBS, purchased from Gibco; Penicillin / Streptomycin, purchased from Gibco; DMEM / F12, purchased from Gibco.

[0033] Example 1. Establishment of culture medium for mammary organoids and / or breast cancer organoids

[0034] Each component was added to DMEM / F12 medium at the following concentrations to obtain medium A for culturing breast cancer organoids:

[0035] Formula 1: estradiol 100nM, hydrocortisone 50μg / ml, R-Spondin 3 (250ng / ml), Neuregulin1 (5nM), FGF7 (5ng / ml), FGF10 (50ng / ml), rh-EGF (5ng / ml), PDGF (platelet-derived growth factor, 5ng / ml), Noggin (100ng / ml), A83-01 (TGF-β type I receptor inhibitor, 500nM), Wnt3a (5nM), Y-27632 (protein kinase p160ROCK inhibitor, 5μM), SB202190 (p38 MAPK inhibitor, 500ng), B27 (10ng / ml), N-Acetylcysteine ​​(5nM), Nicotinamide (10nM), GlutaMax supplement (5nM), fetal bovine serum (FBS, 10%), and Penicillin / Streptomycin (1%).

[0036] Formulation 2: Estradiol 10nM, Hydrocortisone 0.1μg / ml, R-Spondin 3 (100ng / ml), Neuregulin1 (1nM), FGF7 (1ng / ml), FGF10 (10ng / ml), rh-EGF (1ng / ml), PDGF (1ng / ml), Noggin (50ng / ml), A83-01 (100nM), Wnt3a (1nM), Y-27632 (1μM), SB202190 (100ng), B27 (10ng / ml), N-Acetylcysteine ​​(1nM), Nicotinamide (1nM), GlutaMax Supplement (1nM), FBS (10%), Penicillin / Streptomycin (1%).

[0037] Formula 3: Estradiol 1000nM, Hydrocortisone 100μg / ml, R-Spondin 3 (500ng / ml), Neuregulin1 (10nM), FGF7 (10ng / ml), FGF10 (100ng / ml), rh-EGF (10ng / ml), PDGF (10ng / ml), Noggin (500ng / ml), A83-01 (1000nM), Wnt3a (5nM), Y-27632 (10μM), SB202190 (1000ng), B27 (10ng / ml), N-Acetylcysteine ​​(5nM), Nicotinamide (10nM), GlutaMax Supplement (5nM), FBS (10%), Penicillin / Streptomycin (1%).

[0038] Comparative formula 1: The difference from the above formula is that the culture medium does not contain growth factor PDGF. The specific content of each component is as follows: R-Spondin 3 (250ng / ml), Neuregulin 1 (5nM), FGF 7 (5ng / ml), FGF 10 (50ng / ml), rh-EGF (5ng / ml), Noggin (100ng / ml), A83-01 (500nM), Wnt3a (5M), Y-27632 (5μM), SB202190 (500ng), B27 (10ng / ml), N-Acetylcysteine ​​(5nM), Nicotinamide (10nM), GlutaMax (5nM), FBS (10%), Penicillin / Streptomycin (1%), DMEM / F12.

[0039] Comparative formula 2: The difference from the above formula is that the culture medium does not contain FGF 7, FGF 10, rh-EGF, PDGF, and Wnt3a. The specific content of each component is as follows: R-Spondin 3 (250ng / ml), Neuregulin 1 (5nM), Noggin (100ng / ml), A83-01 (500nM), Y-27632 (5μM), SB202190 (500ng), B27 (10ng / ml), N-Acetylcysteine ​​(5nM), Nicotinamide (10nM), GlutaMax (5nM), FBS (10%), Penicillin / Streptomycin (1%), and DMEM / F12.

[0040] Example 2: Method for establishing mammary gland organoids and / or breast cancer organoids

[0041] In this embodiment, breast samples and breast cancer samples were obtained from Chongqing People's Hospital.

[0042] The method for establishing mammary organoids or / and breast cancer organoids using the culture medium of Example 1, the specific steps are as follows:

[0043] 1) Obtaining breast cancer tissue

[0044] After collection, the obtained breast cancer tissue / breast tissue was immersed in 50 ml of DMEM / F12+FBS (10%)+Penicillin / Streptomycin (1%).

[0045] 2) Cultivation of breast cancer organoids

[0046] (1) The obtained breast cancer sample was soaked in 1% Penicillin / Streptomycin for 1 hour, and the soaked breast cancer sample was taken out and the cells were cut into pieces with a disposable scalpel. The cut tissue was about 1 cm×1 cm; the 1 cm×1 cm breast cancer surgical sample was crushed with a grinding rod to prepare a single cell suspension.

[0047] (2) Aspirate the single-cell suspension, filter it with a 200-mesh cell strainer 2-3 times to remove excess residual tissue, add PBS to wash 3 times, add matrigel to resuspend the cells, and inoculate them in a 6-well plate; add culture medium A to the 6-well plate to culture the cells, place it in an incubator at 37°C, change the culture medium every three days, observe the cell morphology, take pictures under an optical microscope, and culture for up to 14 days.

[0048] The results of culturing medium A of formulas 1 to 3 for 1 day, 3 days, 5 days, 7 days, 10 days and 14 days are as follows Figure 1 The statistical results of organoid volume and cell number are shown in Figure 2 As shown, the results showed that the number of cells gradually increased during the culture process, organoids could be formed on the second day of culture, the number of organoid cells increased rapidly on the fourth day of culture, and showed logarithmic growth on the 7th day; at the same time, the organoids gradually became larger during the culture process, reaching a maximum of 900μm on the 14th day of culture.

[0049] The results of the comparison with formula 1 without the important growth factor PDGF are as follows Figure 3 The statistical results of organoid volume and cell number are shown in Figure 4 As shown, compared with the organoids containing PDGF in Formula 1-3, only a small amount of organoids were generated on the third day, and a large number of organoids were not generated until the 10th day. The growth rate of the size and cell number of the organoids generated by Formula 1 was significantly lower than that of Formula 1-3.

[0050] The results of the comparison formula 2 without the important growth factors Wnt3a, FGF 7, FGF 10, PDGF, and rh-EGF are as follows Figure 5 The statistical results of organoid volume and cell number are shown in Figure 6 As shown, very few organoids were generated after 14 days of culture.

[0051] The growth morphology of mammary organoids cultured in medium with formulas 1 to 3 for 3 days, 5 days, and 10 days is shown in the figure. Figure 7 As shown, it is shown that the culture media of formulas 1 to 3 can also be used for the culture of mammary organoids.

[0052] Example 3: HE staining of breast cancer organoids

[0053] Place the organoids in good growth condition into a centrifuge tube, centrifuge at 200g for 10 minutes, discard part of the supernatant, add 2 ml of egg white into the centrifuge tube, shake gently to mix, suspend the cell clusters in the egg white solution, add 80% alcohol with a volume of 5 times the egg white mixture, mix, centrifuge at 200g for 10 minutes, remove the supernatant, and add 10% formaldehyde to fix for 3 hours.

[0054] The tissue samples were immersed in 4% paraformaldehyde solution, embedded in paraffin and sliced ​​after 48 hours.

[0055] HE staining steps are as follows: xylene (Ⅰ) 15min; xylene (Ⅱ) 15min; anhydrous ethanol (Ⅰ) 5min; anhydrous ethanol (Ⅱ) 5min; 95% ethanol 3min; 80% ethanol 2min; 70% ethanol 2min; distilled water 5min; hematoxylin staining solution 6min; tap water rinse 1min; 1% hydrochloric acid ethanol 1-2s; tap water rinse 10-30s; 0.2% ammonia blue 6min; tap water rinse 10-30s; 0.5% eosin staining solution 1-3min; distilled water rinse 1-2s; 70% ethanol 1min; 80% ethanol 1min; 95% ethanol 2min; anhydrous ethanol (Ⅰ) 3min; xylene (Ⅰ) 10min; xylene (Ⅱ) 10min. The results are as follows Figure 8 The results showed that breast cancer organoids formed cell clusters and had a high degree of morphological consistency with breast cancer cells.

[0056] Example 4: CCK-8 toxicity experiment for evaluating drug effects in breast cancer organoids

[0057] 1) Make a standard curve (for determining the specific number of cells)

[0058] (1) First, count the number of cells in the prepared cell suspension using a cell counting plate, and then inoculate the cells into the culture plate.

[0059] (2) Dilute the cells in the culture medium in equal proportions (e.g., 1 / 2 ratio) to form a cell concentration gradient. Generally, 3 to 5 cell concentration gradients are required, and 3 to 6 replicate wells are recommended for each concentration.

[0060] (3) After inoculation, the cells are cultured for 2-4 hours to allow them to adhere to the wall. Then, CCK-8 reagent is added and cultured for a certain period of time, and the OD value is measured to create a standard curve with the number of cells as the horizontal axis (X axis) and the OD value as the vertical axis (Y axis). The number of cells in an unknown sample can be determined based on this standard curve. The premise for using this standard curve is that the experimental conditions must be consistent, so that the number of cells inoculated and the culture time after adding CCK-8 can be determined.

[0061] 2) Cell activity detection

[0062] (1) Inoculate 100 μL / well of the cell suspension in a 96-well plate and place the plate in an incubator at 37°C and 5% CO2 for a period of time.

[0063] (2) Add 10 μL of CCK-8 solution to each well, taking care not to create bubbles in the wells as they will affect the OD reading.

[0064] (3) Incubate the culture plate in an incubator for 1-4 hours.

[0065] (4) Measure the absorbance at 450 nm using an enzyme-labeled instrument.

[0066] (5) If you do not want to measure the OD value temporarily, you can add 10 μL of 0.1M HCl solution or 1% w / v SDS solution to each well and cover the culture plate to avoid light and store at room temperature. If measured within 24 hours, the absorbance will not change.

[0067] 3) Cell proliferation-toxicity assay

[0068] (1) On the seventh day, digestive enzymes were added to digest Matrigel, and 100 μL of cell suspension was prepared in a 96-well plate. The culture plate was placed in an incubator at 37°C and 5% CO2 for pre-incubation for 24 hours.

[0069] (2) Add 10 μL of chemotherapeutic drugs and targeted drugs of different concentrations to the culture plate, including but not limited to 5-fluorouracil, cisplatin, docetaxel, and gemcitabine.

[0070] (3) Incubate the culture plate in an incubator for an appropriate period of time (e.g., 6, 12, 24, or 48 hours).

[0071] (4) Add 10 μL of CCK-8 solution to each well. Be careful not to create bubbles in the wells, as they will affect the OD reading.

[0072] (5) Incubate the culture plate in an incubator for 1-4 hours.

[0073] (6) Measure the absorbance at 450 nm using an enzyme-labeled instrument.

[0074] (7) If you do not want to measure the OD value temporarily, you can add 10 μL of 0.1M HCl solution or 1% w / v SDS solution to each well and cover the culture plate to avoid light and store at room temperature. If measured within 24 hours, the absorbance will not change.

[0075] (8) Finally, the inhibition rates of 5-fluorouracil, cisplatin, docetaxel, gemcitabine, and normal saline on tumor organoids were calculated.

[0076] Note: If the substance to be tested is oxidizing or reducing, you can replace the culture medium with fresh one before adding CCK-8 (remove the culture medium, wash the cells twice with the culture medium, and then add new culture medium) to remove the drug effect. Of course, if the drug effect is relatively small, you can directly subtract the blank absorption after adding the drug to the culture medium without replacing the culture medium. The result is as follows: Fig. 9 As shown, compared with normal saline, 5-fluorouracil, cisplatin, docetaxel, and gemcitabine all have a certain inhibitory effect on breast cancer organoids, and 5-fluorouracil has the highest inhibition rate on tumor organoids, up to 83%.

[0077] The embodiments are only preferred embodiments for fully illustrating the present invention, and the protection scope of the present invention is not limited thereto. Equivalent substitutions or changes made by those skilled in the art based on the present invention are within the protection scope of the present invention. The protection scope of the present invention shall be subject to the claims.

Claims

1. A culture medium for establishing mammary organoids or / and breast cancer organoids, characterized in that: The composition contains the following concentrations: estradiol 1000nM, hydrocortisone 100μg / mL, R-Spondin 3 500ng / mL, Neuregulin 1 10nM, FGF7 10ng / mL, FGF10 100ng / mL, rh-EGF 10ng / mL, PDGF 10ng / mL, Noggin 500ng / mL, A83-01 1000nM, Wnt3a 5nM, Y-27632 10μM, SB202190 1000ng / mL, B27 10ng / mL, N-acetylcysteine ​​5nM, nicotinamide 10nM, GlutaMax 5nM, FBS volume fraction 10%, penicillin / streptomycin double antibody volume fraction 1%.

2. A method for establishing mammary organoids or / and breast cancer organoids based on the culture medium of claim 1, characterized in that: The following steps are involved: (1) preparing the obtained breast tissue or breast cancer tissue into a single cell suspension; the breast tissue or breast cancer tissue is derived from a mammal; the specific process of preparing the breast tissue or breast cancer tissue into a single cell suspension is to cut, grind and filter; the size of the cut tissue is 1 cm×1 cm; the filtration is to filter using a 200-mesh filter; (2) The single cell suspension prepared in step (1) is added to the 3D culture matrix gel to be resuspended, and then inoculated into a culture device, and the culture medium according to claim 1 is added, and the culture is expanded in an incubator, and the culture medium is replaced every three days during the expansion culture.

3. Use of the culture medium according to claim 1 in the culture of breast cancer organoids or / and mammary gland organoids and in the screening of anti-breast cancer drugs.

4. The use according to claim 3, characterized in that: The drugs include chemotherapy drugs and targeted drugs.

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