Culture medium and method for mammary epithelial cells, and use of mammary epithelial cells

Abstract

A culture medium for culturing mammary epithelial cells, comprising β-estradiol, insulin-like growth factor 1, basic fibroblast growth factor, tumor necrosis factor-α, and optionally fibroblast growth factor 10. A method for culturing mammary epithelial cells using said culture medium, as well as a method and the use of cells obtained using said culture method in evaluating the efficacy of drugs or screening drugs.

Description

[Technical field]

[0001] The present invention relates to a culture medium for in vitro culture of epithelial cells, particularly mammary epithelial cells, and a culture method for culturing said cells or organoids comprising said cells.The present invention also relates to the use of the cell progeny or organoids cultured by the culture medium and culture method of the present invention in drug efficacy evaluation and screening, toxicity determination, and regenerative medicine. [Background technology]

[0002] In vitro culture of human mammary epithelial cells is crucial for studying the mechanisms of normal breast development, as well as the occurrence and progression of breast tumors. However, many challenges remain in achieving sustainable culture of human mammary epithelial cells. Currently, two regenerative techniques have been reported that can be used for in vitro culture of human mammary epithelial cells. One is the conditional reprogramming technique developed at Georgetown University in the United States, and the other is the organoid technique developed at the Royal Netherlands Academy of Arts and Sciences. However, there are certain problems in the practical application of these two techniques: in the conditional reprogramming technique, the patient's autologous tumor cells need to be co-cultured with mouse-derived feeder cells, which may interfere with the analysis results of the tumor cells. Although the culture medium used in the organoid technique has stem cell niche factors as its core component, this culture technique requires a large amount of niche factors, is expensive, and has a long culture cycle, making it difficult to operate. Therefore, neither of these two techniques has actually been promoted on a large scale in the fields of basic research and new drug development.

[0003] In Patent Document 1, the present inventors have described a culture medium and a culture method for in vitro culture of mammary epithelial cells without feeder cells, at manageable costs and convenient operation. This is the first 2D culture system for mammary epithelial stem cells in vitro that does not require feeder cells and Wnt agonists such as Wnt proteins and proteins of the R-spondin family as culture components. In this culture system, continuous proliferation of mammary epithelial cells can be maintained in vitro for at least one month. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] International Publication No. 2021 / 088119 Summary of the Invention

[0005] Based on the original invention, it was unexpectedly discovered that by applying an additive such as tumor necrosis factor to cultures of mammary epithelial cells, a more rapid promotion of continuous proliferation of the mammary epithelial cells can be achieved. The present invention provides a culture medium and a culture method for culturing mammary epithelial cells. The culture medium comprises β-estradiol, insulin-like growth factor 1, basic fibroblast growth factor, tumor necrosis factor-α, and optionally fibroblast growth factor 10.

[0006] In an embodiment of the present invention, the amount of each component in the culture medium of the present invention satisfies the following conditions: The concentration of β-estradiol is 5 nM to 50 nM, more preferably 5 nM to 10 nM; the concentration of insulin-like growth factor 1 is 10 ng / ml to 200 ng / ml, more preferably 20 ng / ml to 100 ng / ml; The concentration of basic fibroblast growth factor is 10 ng / ml to 200 ng / ml, more preferably 20 ng / ml to 100 ng / ml; A concentration of tumor necrosis factor-α is 2ng / ml to 100ng / ml, more preferably 5ng / ml to 50ng / ml; and Fibroblast growth factor 10 is optionally added at a concentration of 0 ng / ml to 50 ng / ml; Satisfy one or more or all of the following.

[0007] In an embodiment of the invention, the culture medium of the invention further comprises amphiregulin, epidermal growth factor, insulin, B27, ROCK inhibitor Y27632, neuregulin 1, fibroblast growth factor 7, TGFβ type I receptor inhibitor A8301, and GlutaMAX-I.

[0008] Here, in a preferred embodiment, the amount of amphiregulin is 10 ng / ml to 100 ng / ml, the amount of epidermal growth factor is 2.5 ng / ml to 20 ng / ml, the amount of insulin is 1 μg / ml to 10 μg / ml, B27 is added at a volume ratio of 1:25 to 1:100, the amount of Y27632 is 5 μM to 15 μM, the amount of neuregulin 1 is 5 nM to 20 nM, the amount of fibroblast growth factor 7 is 2.5 ng / ml to 20 ng / ml, the amount of A8301 is 100 nM to 500 nM, and GlutaMAX-I is added at a volume ratio of 1:50 to 1:200.

[0009] In an embodiment of the invention, the culture medium further comprises an initial culture medium selected from the group consisting of DMEM / F12, DMEM, F12 or RPMI-1640, and one or more antibiotics selected from the group consisting of streptomycin / penicillin, amphotericin B and Primocin.

[0010] In a preferred embodiment, if streptomycin / penicillin is used as the antibiotic(s), the concentration range for streptomycin is 25μg / mL to 400μg / mL and the concentration range for penicillin is 25U / mL to 400U / mL. If amphotericin B is used as the antibiotic, the concentration range is 0.25μg / mL to 4μg / mL. If Primocin is used as the antibiotic, the concentration range is 25μg / mL to 400μg / mL.

[0011] A second aspect of the present invention provides a culture method for mammary epithelial cells, comprising the following steps: (1) preparing a culture medium for the mammary epithelial cells of the present invention; (2) coating a culture vessel with an extracellular matrix gel; and (3) inoculating primary mammary epithelial cells into the coated culture vessel and culturing the cells using the culture medium for the mammary epithelial cells of the present invention.

[0012] Here, the extracellular matrix gel used in the culture method is a growth factor reduced-type extracellular matrix gel, for example, commercially available Matrigel (Corning: 354230) or BME (Trevigen: 3533-010-02). More specifically, the extracellular matrix gel is diluted with a serum-free culture medium, which may be the initial culture medium in the present invention, for example, DMEM / F12 (Corning: R10-092-CV). The dilution ratio of the extracellular matrix gel is 1:20 to 1:400, preferably 1:50 to 1:200. The coating method is to add the diluted extracellular matrix gel to the culture vessel so that the bottom of the culture vessel is completely covered, and leave it for more than 30 minutes, preferably at 37°C, and preferably for 30 to 60 minutes to coat it. After completion of coating, the excess diluted extracellular matrix gel is aspirated and discarded, and the culture vessel is ready for use.

[0013] The human mammary epithelial cells may be breast cancer tumor cells, normal mammary epithelial cells, or mammary epithelial stem cells. For example, the tissue sample is taken within 30 minutes of surgical resection or biopsy on the patient. More specifically, in a sterile environment, a tissue sample from a non-necrotic area is taken from 0.5 cm 3 Cut out the above volume, place it in pre-cooled antibiotic-containing DMEM / F12 culture medium, and transport it to the laboratory on ice. For example, use DMEM / F12 culture medium containing 50 U / mL to 200 U / mL (e.g., 100 U / mL) penicillin and 50 μg / mL to 200 μg / mL (e.g., 100 μg / mL) streptomycin for ice-cooled transportation.

[0014] In a biosafety cabinet, the tissue sample is transferred to a cell culture dish and then rinsed with transport fluid. Blood cells on the surface of the tissue sample are washed away, and unnecessary tissues such as skin and fascia on the surface of the tissue sample are removed.

[0015] Transfer the rinsed tissue sample to another new culture dish and add 5 mL to 25 mL of transport solution. Cut the tissue sample into pieces with a diameter of 1 mm using a sterile scalpel blade and tweezers. 3 Divide into smaller tissue sections.

[0016] Transfer the tissue sample slices to a centrifuge tube and centrifuge in a tabletop centrifuge at 1000 rpm or higher for 3 to 10 minutes. After carefully removing the supernatant from the centrifuge tube using a pipettor, resuspend the precipitate in 5 mL to 25 mL of serum-free DMEM / F12 culture medium containing collagenase II (0.5 mg / mL to 5 mg / mL, e.g., 1 mg / mL) and collagenase IV (0.5 mg / mL to 5 mg / mL, e.g., 1 mg / mL), place in a 37°C thermostatic shaker, and perform shaking digestion for at least 30 minutes (digestion time depends on the sample size; if the sample is more than 1 g, extend the digestion time to 1.5 hours to 2 hours), and then centrifuge in a tabletop centrifuge at 300 g / min or higher for 3 to 10 minutes. Discard the supernatant and resuspend the digested tissue cells in, for example, 5 mL to 25 mL of DMEM / F12 culture medium containing 10% fetal bovine serum, grind, and sieve, for example, through a cell sieve with a pore size of 100 μm. Collect the sieved cell suspension and place it into a centrifuge tube. Count the cells using a hemocytometer.

[0017] The cell suspension is then centrifuged in a centrifuge at at least 300 g / min for 3-10 minutes. The supernatant is discarded and the pellet is resuspended in the culture medium of the present invention and then plated at 1×10 cells per well onto the coated culture vessel for culture. 4 pieces~1×10 6 Inoculate at a density of .

[0018] A third aspect of the present invention provides a method for evaluating or screening drugs for treating breast disease, comprising obtaining expanded progeny mammary epithelial cells using the culture medium and culture method of the present invention, and applying the obtained cells to drug efficacy evaluation and screening, in particular to in vitro efficacy evaluation and screening of anti-tumor drugs.

[0019] Preferably, the method for evaluating or screening a drug for treating a breast disease comprises the following steps: (1) obtaining primary mammary epithelial cells and culturing them using the culture method of the present invention; (2) selecting a drug to be tested and preparing different concentration gradients; (3) adding the drug at different concentrations prepared in step (2) to the mammary epithelial cells cultured in step (1); (4) detecting cell viability; Includes.

[0020] Beneficial effects of the present invention include the following: (1) Rapid expansion of mammary epithelial cells can be achieved in a short period of time, and sufficient numbers of cells can be obtained within an effective time frame for basic research and drug screening applications. (2) Mammary epithelial cells obtained by in vitro culture using the culture medium and culture method of the present invention can maintain the heterogeneity of the patient from which the cells are derived, and can be applied in the field of regenerative medicine; (3) The cultured mammary epithelial cells are not interfered with by cells such as fibroblasts, and purified mammary epithelial cells and their progeny can be obtained. (4) the culture medium does not contain any uncertain components such as serum, and therefore is not affected by the quality and quantity of serum from different batches; and (5) In the present technology, there is no need to add costly stem cell niche factors such as R-spondin and Wnt family components to culture mammary epithelial cells, and compared with the culture medium disclosed in Patent Document 1, in the culture medium of the present invention, a large number of monolayer epithelial cells can be obtained more rapidly, and the cycle of expanding to obtain the same number of cells is shorter, which are suitable for application in the fields of drug efficacy evaluation, screening and toxicity testing, for example, high-throughput screening of new candidate compounds, and provide in vitro high-throughput drug susceptibility functional testing for patients.

[0021] The culture medium of the present embodiment can be used to culture mammary epithelial cells derived from humans or other mammals, or tissues containing at least one of these cells, to obtain expanded corresponding mammary epithelial cell progeny.

[0022] Furthermore, the cells obtained by the culture method of this embodiment can be applied to regenerative medicine, toxicity testing, basic medical research on mammary epithelial cells, screening of drug response, determination of in vitro metabolic stability and metabolic profile of drugs, and development of new drugs for breast diseases. [Brief description of the drawings]

[0023] [Figure 1-1] 1A-1F are graphs depicting the effect of different concentrations of additive components in culture medium on the proliferation of primary mammary epithelial cells in vitro. [Figure 1-2] FIG. 1G is a graph showing the effect of different concentrations of additive components in culture medium on the proliferation of primary mammary epithelial cells in vitro. [Diagram 2] Figure 2A shows the results of fluorescent staining of the nuclei of primary mammary epithelial cells obtained by culturing primary mammary tumor cells isolated from clinical tissue samples of breast cancer in parallel using the culture medium for mammary epithelial cells of the present invention and the control culture medium until passage 3, and then continuing the culture for 14 days (100x microscope). Figure 2B shows the results of fluorescent labeling (DAPI) of the cell nuclei of cells obtained by culturing primary cells derived from three different breast tumor patients in parallel using the same culture method as in Figure 2A, and statistical analysis. "***" indicates P<0.001. [Diagram 3] 3A and 3B are a comparison diagram and a curve graph of culture days versus population doubling number, respectively, showing the effect of continuous culture using the culture medium of the present invention and a control culture medium on an example of primary mammary epithelial cells. [Figure 4] 4A and 4B are a comparison diagram and a curve graph of culture days versus population doubling number, respectively, showing the effect of continuous culture using the culture medium of the present invention and a control culture medium on another example of primary mammary epithelial cells. [Diagram 5]This figure shows the results of DAPI fluorescent labeling, immunofluorescence staining using a biomarker specific to luminal epithelial cells (CK8) and a biomarker specific to myoepithelial cells (CK14), and multichannel fluorescent signal overlap imaging (MERGE) of mammary epithelial cells obtained by culturing primary breast cancer cells isolated from clinical tissue samples of breast cancer in parallel using a culture medium for mammary epithelial cells of the present invention and a control culture medium up to the third passage and then continuing the culture for 14 days (under a 100x microscope). [Figure 6] 6A-6F are dose-effect curves of various drugs on breast tumor cells cultured using the culture medium for breast epithelial cells of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0024] Example 1 Isolation of human primary mammary epithelial cells and optimization of culture medium for mammary epithelial cells

[0025] (1) Isolation of primary human mammary epithelial cells For transport and washing of samples, a commercially available penicillin-streptomycin double antibody solution (Corning, containing 10,000 U / ml penicillin and 10 mg / ml streptomycin) was added to DMEM / F12 culture medium (manufactured by Corning) at a volume ratio of 2%. This is hereinafter referred to as the "transport solution."

[0026] Breast tumor tissue samples 1#, 2#, 3#, 4#, and 5# were derived from surgically removed cancer tissue samples from five breast tumor patients who provided informed consent. One of these samples (1#) is used in the following description. The above tissue samples were collected within 30 minutes after surgical resection from the patients. More specifically, tissue samples of 0.5 cm in volume were extracted from non-necrotic areas in a sterile environment. 3 The above tissue samples were excised, placed in pre-chilled transport fluid, and shipped on ice to the laboratory.

[0027] In a biosafety cabinet, the tissue sample (1#) was transferred to a 100 mm cell culture dish. The tissue sample was rinsed with transport solution. Blood cells on the surface of the tissue sample were washed away. Unwanted tissues such as skin and fascia on the surface of the tissue sample were removed.

[0028] Transfer the rinsed tissue sample to another new 100 mm culture dish, add 10 mL of transport solution, and use a sterile scalpel blade and tweezers to cut the tissue sample into pieces 1 mm in diameter. 3 The tissue was divided into sections of less than 1 mm.

[0029] The tissue sample slices were transferred to a 50 mL centrifuge tube and centrifuged at 1200 rpm for 5 minutes using a tabletop centrifuge. After carefully removing the supernatant from the centrifuge tube using a pipettor, the precipitate was resuspended in 10 mL of serum-free DMEM / F12 culture medium containing collagenase II (1 mg / mL) (Sigma) and collagenase IV (1 mg / mL) (Sigma), placed in a 37°C thermostatic shaker for 30 to 90 minutes of shaking digestion, and then centrifuged at 350 g / min for 5 minutes in a tabletop centrifuge. The supernatant was discarded, and the digested tissue cells were resuspended in 10 mL of DMEM / F12 culture medium containing 10% fetal bovine serum (Gibco), ground, and sieved. The pore size of the cell sieve was, for example, 100 μm. The sieved cell suspension was collected and placed in a 50 mL centrifuge tube. The cells were counted using a hemocytometer.

[0030] The cell suspension was then centrifuged at 350 g / min for 5 minutes in a centrifuge, and after discarding the supernatant, the pellet was resuspended in the basal medium described below.

[0031] Another four breast tumor tissue samples were isolated following the same process as above.

[0032] (2) Coating of cell culture plates Extracellular matrix gel (Matrigel, Corning) was diluted with DMEM / F12 at a ratio of 1:50 to prepare a diluted extracellular matrix gel. 250 μl / well of the diluted extracellular matrix gel was added to a 24-well culture plate so that the bottom of the culture plate well was completely covered. After leaving it in a 37° C. incubator for 1 hour, the diluted extracellular matrix gel was removed to obtain a culture plate coated with extracellular matrix gel.

[0033] (3) Screening of supplemental factors for culture medium of primary mammary epithelial cells First, a basic culture medium was prepared based on Patent Document 1. GlutaMAX-I (manufactured by Thermo Fisher Scientific) was added to a commercially available DMEM / F-12 culture medium at a concentration specified in the instruction manual (1:100 dilution), human insulin (Sigma) was added at a final concentration of 10 μg / ml, ROCK inhibitor Y27632 (Sigma) was added at a final concentration of 10 μM, penicillin-streptomycin (Thermo Fisher Scientific) was added at a 1:100 dilution, human amphiregulin (manufactured by R&D Systems) was added at a final concentration of 20 ng / ml, epidermal growth factor (EGF, manufactured by Peprotech) was added at a final concentration of 10 ng / ml, B27 (Thermo Fisher Scientific) was added at a 1:50 dilution, human neuregulin 1 (Peprotech) was added at a final concentration of 10 nM, and fibroblast growth factor 7 (FGF7, manufactured by R&D Systems) was added at a final concentration of 10 nM. A basal culture medium for primary mammary epithelial cells was prepared by adding TGFβ1 inhibitor A8301 (manufactured by MCE) to a final concentration of 500 nM. This is hereinafter referred to as the "basal medium."

[0034] Then, various types of additives (Table 1) were added to the base medium to prepare culture media for mammary epithelial cells containing different additives. The culture media with different components were added to the 24-well plates coated with extracellular matrix gel in a volume of 500 μl / well. Three replicates were prepared for each culture medium formulation. Breast cancer cells (1#) isolated from the mammary tumor tissues in Example (1) were added to the 24-well culture plates coated with extracellular matrix gel at 3×10 cells per well. 4 The cells were inoculated at a cell density of 1000 x 1000 and cultured at 37°C and 5% CO2 concentration using different culture medium formulations. After the start of culture, the culture medium was changed every 3 days. After 14 days of culture, the cells were counted. The basal medium without any of the additives shown in Table 1 was used as the experimental control.

[0035] Tumor cells isolated from the breast tumor tissue samples of the remaining four cases were cultured and counted in the same manner as above.

[0036] The statistical results are shown in Table 1.

[0037] [Table 1]

[0038] Here, "+" indicates that compared to the basal medium, the culture medium supplemented with the additive(s) has an effect of promoting the proliferation of at least three cases of primary breast cancer cells isolated from breast tumor tissue. The number of "+" indicates the degree of proliferation promotion. "-" indicates that the culture medium supplemented with the additive(s) has an effect of inhibiting the proliferation of at least two cases of primary breast cancer cells isolated from breast tumor tissue. "○" indicates that the culture medium supplemented with the additive(s) has no significant effect on the proliferation of at least three cases of primary breast cancer cells isolated from breast tumor tissue.

[0039] The results showed that several additives, including insulin-like growth factor 1, basic fibroblast growth factor, fibroblast growth factor 10, and tumor necrosis factor α, could promote the growth of at least three primary breast tumor cells isolated from breast tumor tissues, and among these additives, the effects of basic fibroblast growth factor and tumor necrosis factor α were stronger. When all of the above factors were added to the basal medium, the degree of growth promotion was significantly increased.

[0040] (4) Optimization of the concentration of additives in culture media for human mammary epithelial cells Seven different culture medium formulations were also prepared: Formulation 1: Base medium supplemented with 1:100 diluted MEM-NEAA, 1 mM sodium pyruvate, 50 ng / ml IGF1, 20 ng / ml bFGF, 10 ng / ml TNF-α, and 20 ng / ml FGF10; Formulation 2: basal medium supplemented with 10 nM β-estradiol, 1 mM sodium pyruvate, 50 ng / ml IGF1, 20 ng / ml bFGF, 10 ng / ml TNF-α and 20 ng / ml FGF10; Formulation 3: basal medium supplemented with 10 nM β-estradiol, 1:100 diluted MEM-NEAA, 50 ng / ml IGF1, 20 ng / ml bFGF, and 10 ng / ml TNF-α and 20 ng / ml FGF10; Formulation 4: basal medium supplemented with 10 nM β-estradiol, 1:100 diluted MEM-NEAA, 1 mM sodium pyruvate, 20 ng / ml bFGF, and 10 ng / ml TNF-α and 20 ng / ml FGF10; Formulation 5: basal medium supplemented with 10 nM β-estradiol, 1:100 diluted MEM-NEAA, 1 mM sodium pyruvate, 50 ng / ml IGF1, and 10 ng / ml TNF-α and 20 ng / ml FGF10; Formulation 6: Basal medium supplemented with 10 nM β-estradiol, 1:100 diluted MEM-NEAA, 1 mM sodium pyruvate, 50 ng / ml IGF1, 20 ng / ml bFGF, and 20 ng / ml FGF10; Formulation 7: Basal medium supplemented with 10 nM β-estradiol, 1:100 diluted MEM-NEAA, 1 mM sodium pyruvate, 50 ng / ml IGF1, 20 ng / ml bFGF and 10 ng / ml TNF-α.

[0041] Then, β-estradiol (Sigma) was added to formulation 1 to prepare culture media for human mammary epithelial cells containing β-estradiol at final concentrations of 5 nM, 10 nM, 50 nM, and 100 nM, and each culture medium was added to a 96-well plate coated with Matrigel in a volume of 100 μl / well. Three replicates were used for each concentration. Primary breast cancer cells (2#) isolated from breast tumor tissues in Example (1) were seeded at a density of 500 cells per well in a 96-well culture plate coated with Matrigel and cultured in formulation 1 containing different concentrations of β-estradiol at 37°C and 5% CO2. After the start of culture, the culture medium was replaced every 3 days. On the 14th day of culture, the cells were counted. Formulation 1 was used as the experimental control, i.e., the content of β-estradiol in formulation 1 was 0 nM. The results are shown in Figure 1A.

[0042] Non-essential amino acids (MEM-NEAA) additive (Thermo Fisher) was added to formulation 2 at dilution ratios of 1:200, 1:100, 1:50, and 1:20 to prepare culture media for human mammary epithelial cells containing different concentrations of non-essential amino acids. The culture media of the different formulations were added to a Matrigel-coated 96-well plate at a volume of 100 μl / well. Three replicates were used for each concentration. Primary cells were cultured and counted in the same manner as above. Formulation 2 was used as the experimental control, i.e., the content of non-essential amino acids in formulation 2 was 0. The results are shown in Figure 1B.

[0043] Similarly, sodium pyruvate (Thermo Fisher) was added to formulation 3 to prepare culture media for human mammary epithelial cells containing sodium pyruvate at final concentrations of 0.5 mM, 1 mM, 2 mM, and 4 mM. The culture media of different formulations were added to a Matrigel-coated 96-well plate in a volume of 100 μl / well. Three replicates were used for each concentration. Primary cells were cultured and counted in the same manner as above. Formulation 3 was used as the experimental control, i.e., the content of sodium pyruvate in formulation 3 was 0. The results are shown in Figure 1C.

[0044] Then, insulin-like growth factor 1 (IGF1, manufactured by R&D) was added to formulation 4 to prepare culture media for human mammary epithelial cells containing IGF1 at final concentrations of 10ng / ml, 20ng / ml, 50ng / ml, 100ng / ml, and 200ng / ml, and the culture media containing different concentrations of IGF1 were added to a 96-well plate coated with Matrigel in a volume of 100μl / well. Three replicates were used for each concentration. Primary cells were cultured and counted in the same manner as above. Formulation 4 was used as the experimental control, i.e., the content of IGF1 in formulation 4 was 0. The results are shown in Figure 1D.

[0045] Similarly, culture media for human mammary epithelial cells containing different concentrations of basic fibroblast growth factor (bFGF, manufactured by R&D) were prepared using Formula 5, with the final concentrations of bFGF being 5ng / ml, 10ng / ml, 20ng / ml, 50ng / ml, 100ng / ml, and 200ng / ml, respectively. Primary cells were cultured and counted in the same manner as above. Formula 5 was used as the experimental control, i.e., the content of bFGF in Formula 5 was 0. The results are shown in Figure 1E.

[0046] Then, culture media for human mammary epithelial cells containing different concentrations of tumor necrosis factor-α (TNF-α, Novoprotein) were prepared using Formula 6, with the final concentrations of TNF-α being 2ng / ml, 5ng / ml, 10ng / ml, 50ng / ml, and 100ng / ml, respectively. Primary cells were cultured and counted in the same manner as above. Formula 6 was used as the experimental control, i.e., the content of TNF-α in Formula 6 was 0. The results are shown in Figure 1F.

[0047] Finally, culture media for human mammary epithelial cells containing different concentrations of fibroblast growth factor 10 (FGF10, manufactured by R&D) were prepared using Formula 7, with the final concentrations of FGF10 being 20ng / ml, 50ng / ml, 100ng / ml, and 200ng / ml, respectively. Primary cells were cultured and counted in the same manner as above. Formula 7 was used as the experimental control, i.e., the content of FGF10 in Formula 7 was 0. The results are shown in Figure 1G.

[0048] The results show that different concentrations of additives had a dose-dependent proliferation-promoting effect on the proliferation of primary breast cancer cells in vitro within a certain concentration range, where β-estradiol, IGF1, bFGF, and TNF-α began to show obvious proliferation effects when added at low concentrations. Moreover, the preferred concentration of β-estradiol was 5nM-50nM, more preferably 5nM-10nM, the preferred concentration of IGF1 was 10ng / ml-200ng / ml, more preferably 20ng / ml-100ng / ml, the preferred concentration of bFGF was 10ng / ml-200ng / ml, more preferably 20ng / ml-100ng / ml, and the preferred concentration of TNF-α was 2ng / ml-100ng / ml, more preferably 5ng / ml-50ng / ml. Furthermore, the additive FGF10 also had a dose-dependent proliferation-promoting effect on the proliferation of primary breast cancer cells in vitro within a certain concentration range, and the preferred concentration of the additive was 0 ng / ml to 50 ng / ml.

[0049] (5) Test on the proliferation-promoting effect of culture medium for human mammary epithelial cells Two different formulations of media were further prepared as follows: 1. Control culture medium: The preparation procedure was based on Patent Document 1. That is, a culture medium containing the following components was prepared: DMEM / F-12 culture medium (Corning) + GlutaMAX-I (Thermo Fisher SCIENTIFIC) at a volume ratio of 1:100 + 10 μg / ml human insulin (Sigma) + 10 μM Y27632 (Sigma) + 1:100 volume ratio penicillin-streptomycin (Thermo Fisher SCIENTIFIC) + 20 ng / ml human amphiregulin (R&D Systems) + 10 ng / ml EGF (Peprotech) + 1:50 volume ratio B27 (Thermo Fisher SCIENTIFIC) + 10 nM human neuregulin 1 (Peprotech) + 10 ng / ml FGF7 (R&D Systems) + 500 nM A8301 (MCE) + 500 nM SB202190; 2. Complete culture medium (hereinafter referred to as "culture medium of the present invention"): 10 nM β-estradiol, 20 ng / ml bFGF, 50 ng / ml IGF1, and 10 ng / ml TNF-α were added to the basal medium.

[0050] The growth effects of the control culture medium and the culture medium of the present invention on primary breast cancer cells in vitro were compared. The control culture medium and the culture medium of the present invention were added to a 24-well plate coated with Matrigel at a volume of 500 μl / well. Three replicates per group were used.

[0051] According to the method of Example 1(1), breast cancer cells (1#-3#) were isolated from breast tumor tissues and plated in a 24-well culture plate coated with Matrigel at 3 × 10 cells per well. 4The cells were seeded at a cell density of 1000 μg / mL and cultured at 37°C and 5% CO2 using two different culture medium formulations. The cells were subcultured at equal seeding densities until passage 3, and then cultured until day 14. The breast cancer cells cultured using different culture medium formulations were subjected to nuclear fluorescent staining and counted. Specifically, the cells of each group were rinsed twice with PBS buffer, fixed with 4% paraformaldehyde for 15 min, and then incubated with TBST (TBS (manufactured by Sangon Biotech (Shanghai)) + 0.1% Tween 20) containing 1% BSA (Sangon Biotech (Shanghai)) and 1% Triton X-100 (Sangon Biotech (Shanghai)) at room temperature for 1 h, and then rinsed three times with TBST buffer for 3 min each. The TBST solution was removed. The cells were incubated with 1 μg / mL DAPI dye (Sigma) for 10 min. The cells were rinsed once with PBS. Random-field photographs were taken under a 100× fluorescent microscope after mounting the coverslips with a drop of mounting medium (Thermo Fisher Scientific).

[0052] Representative results are shown in Figures 2A and 2B. Figure 2A shows that 2# breast cancer cells were continuously cultured with the control culture medium and the culture medium of the present invention until the third passage, and then 3 x 10 cells were cultured per well. 4 2A and 2B are micrographs of cells cultured at a density of 1# for 14 days. FIG. 2B shows the statistics of DAPI counting results for breast cancer cells (1#-3#) derived from different patients using the same procedure as 2#. The vertical axis shows the statistics of counting results for DAPI staining under random fields. As shown in FIG. 2A and FIG. 2B, compared with the control culture medium, the number of cells cultured to the third passage using the culture medium of the present invention can be increased by about 5.2-fold within 14 days.

[0053] [Example 2] Continuous in vitro culture of human mammary epithelial cells and comparison with existing culture methods (1) Culture of primary mammary epithelial cells derived from human breast tumor tissue Using the same method as in Example 1(1), primary breast cancer cells (6#, 7#) were isolated from cancer tissues of two breast tumor patients. The isolated breast cancer cells were then counted using a hemocytometer, and the 6# cells were then plated at the same density (2×10 cells per well) on two 6-well plates coated with Matrigel (Corning). 5 The coating method was as follows: Matrigel was diluted with DMEM / F12 culture medium at a ratio of 1:50 to prepare a diluted extracellular matrix solution, and 1.5 ml / well of the diluted extracellular matrix gel solution was added to a 6-well culture plate so that the bottom of the culture plate well was completely covered. After leaving it in a 37°C incubator for 1 hour, the diluted extracellular matrix gel solution was removed to obtain a culture plate coated with extracellular matrix.

[0054] The control culture medium prepared in (5) of Example 1 and the culture medium of the present invention were each added at 3 mL / well to two culture wells of a 6-well culture plate coated with extracellular matrix, and cultured at 37° C. and 5% CO2 concentration. After the start of culture, the culture medium was replaced every 3 days.

[0055] Freshly isolated primary breast cancer cells #7 were subjected to the same procedure as #6.

[0056] (2) Comparison of the effects of in vitro continuous culture using different culture media for human mammary epithelial cells Human breast cancer cells in the culture plate were grown until approximately 80% of the bottom area was covered, and the supernatant of the culture medium in the original 6-well plate was discarded. The cells were digested by adding 1 mL of 0.05% trypsin (Thermo Fisher: 25300062) and incubating at 37°C for 10 to 20 minutes. Once the cells were completely digested, the digested cells were resuspended in 5 mL of DMEM / F12 culture medium containing 10% (v / v) fetal bovine serum, 100 U / mL penicillin and 100 μg / mL streptomycin, collected, placed in a centrifuge tube, and centrifuged at 300 g / min for 5 minutes. The cell pellet after centrifugation was resuspended in control culture medium or the culture medium of the present invention, and cell counting was performed on the cell suspension using a counting chamber. The cells of each group were seeded in another 12-well culture plate coated with Matrigel at a passage ratio of 1:10, and the culture was continued.

[0057] After passage, the cells were grown again until they covered about 80% of the bottom area of ​​the culture plate, and the cultured cells were digested again, harvested, and counted by the above-mentioned procedure. The cells were again inoculated at a ratio of 1:10 and continuously cultured.

[0058] The same procedure as in 6# was repeated for 7# primary breast cancer cells.

[0059] FIG. 3A shows cell photographs taken under a 100× phase contrast microscope of 6# cells cultured to passage 2 and passage 5 using the control culture medium and the culture medium of the present invention, respectively.

[0060] Figure 3B is a continuous growth curve of 6# cells under the culture conditions of the culture medium of the present invention and the control culture medium, plotted using GraphPad Prism 7.0 software. The horizontal axis is the number of days in culture, and the vertical axis is the number of cell population doublings.

[0061] The formula for calculating the population doublings of primary mammary epithelial cells under different technical culture conditions is: Population doubling number = [log(N / X0)] / log2 where N is the number of cells at passage and X0 is the number of cells at the initial inoculation (see Greenwood et al., Environ Mol Mutagen 2004, 43 (1): 36-44).

[0062] Figure 4A shows cell photographs of 7# cells cultured to the second and fourth passages after continuous culture according to the same operation steps using the control culture medium and the culture medium of the present invention, respectively. Figure 4B shows a curve chart of the number of culture days-cell population doublings of 7# cells under the culture conditions of the culture medium of the present invention and the control culture medium, plotted using GraphPad Prism 7.0 software.

[0063] From Figures 3A, 3B and 4A, 4B, it can be seen that breast cancer cells cultured using the culture medium of the present invention can proliferate sustainably, and the proliferation rate is significantly better than that in the known control culture medium.

[0064] [Example 3] Identification of immune markers in mammary epithelial cells (1) A control culture medium and a culture medium of the present invention were prepared as described in Example 1(5). (2) Cancer tissues the size of a soybean grain were obtained from clinical surgical resection specimens of breast cancer patients, and primary breast cancer cells (3#) were isolated and obtained using the same method as in (1) of Example 1. The primary breast cancer cells (3#) were cultured up to the third passage using the methods in (1) and (2) of Example 2 in parallel. (3) The expression of important cancer-related biomarkers in human breast cancer cells was detected by immunofluorescence.

[0065] The primary antibodies used in this experiment were CK8 (manufactured by Abcam) and CK14 (Abcam). For CK8, anti-mouse IgG (H+L), F(ab')2 fragment (Alexa Fluor™ 488 Conjugate) (manufactured by Cell Signaling Technology) was used as the secondary antibody, and for CK14, anti-rabbit IgG (H+L), F(ab')2 fragment (Alexa Fluor™ 594 Conjugate) (Cell Signaling Technology) was used as the secondary antibody. Here, CK8 is an important biomarker for breast cancer and is generally expressed on luminal epithelial cells of breast tumors, and CK14 is recognized as an important biomarker for myoepithelial cells of breast tumors. In clinical practice, CK8 and CK14 are often used in the differential diagnosis of breast cancer.

[0066] Specifically, the supernatant of the culture medium in the original 6-well plate was discarded, 1 mL of 0.05% trypsin (Thermo Fisher) was added to digest the cells, and after incubation at 37°C for 15 minutes, the digested cells were resuspended in 5 mL of DMEM / F12 culture medium containing 10% (v / v) fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin, collected into a centrifuge tube, and centrifuged at 300 g / min for 5 minutes. After centrifugation, the cell pellets of each group were resuspended using the culture medium of the present invention and the known control culture medium, respectively. Cell counting was performed on the cell suspension using a counting chamber. The cells were plated on Matrigel-coated coverslips at 4 × 10 cells per coverslip. 4 The cells were seeded at a density of 1000 cells / well. The coating method was the same as in Example 1 (2). Each group of cells was cultured using the culture medium of the present invention and a known control culture medium, respectively, so that the cells grew on the cover glass.

[0067] After 14 days of culture, the cells were rinsed twice with PBS buffer, fixed with 4% paraformaldehyde for 15 min, then incubated with TBST (TBS + 0.1% Tween 20) containing 1% BSA (Sangon Biotech (Shanghai)) and 1% Triton X-100 for 1 h at room temperature, and rinsed three times with TBST buffer for 3 min each time. After removing the TBST solution, 50 μL of the primary antibody dilution solution (CK8 antibody diluted 1:100; CK14 antibody diluted 1:1000) was added to the slide, incubated at 4 °C for 12-16 h, and then rinsed three times with PBS for 3 min each time. Secondary antibody anti-mouse IgG (H + L), F(ab')2 fragment (Alexa Fluor™ 488 Conjugate) (8 μg / ml) was added, incubated at room temperature for 60 min, and rinsed three times with PBS for 3 min each time. Secondary antibody anti-rabbit IgG(H+L), F(ab')2 fragment (Alexa Fluor™ 594 Conjugate) (8 μg / ml) was then added and incubated for 60 min at room temperature and rinsed three times with PBS for 3 min each time. Cells were incubated with 1 μg / mL DAPI dye (Sigma) for 10 min and rinsed once with PBS. After mounting with a drop of mounting medium (Thermo Fisher Scientific), pictures were taken under a fluorescent microscope at 100× magnification.

[0068] The results are shown in Figure 5, which shows the results of immunofluorescence staining with a biomarker specific for luminal epithelial cells (CK8) and myoepithelial cells (CK14), and further shows the location and number of cell nuclei using DAPI fluorescent labeling, and the imaging results generated by overlapping the three fluorescent staining labels CK8, CK14, and DAPI using MERGE.

[0069] According to FIG. 5, by using the culture medium and culture method of the present invention, a large number of cells highly expressing CK8 and CK14 proteins, which are breast cancer cell-specific biomarkers, can be cultured, while only a small number of breast cancer cells can be obtained from primary breast cancer cells derived from the same sample cultured using a known control culture medium, thereby indicating that the culture technique of the present invention can achieve the effect of efficiently culturing breast cancer cells.

[0070] [Example 4] Drug sensitivity testing of breast cancer cells in vitro In the following, a surgically resected sample from a breast tumor patient is used as an example to show that breast cancer cells cultured from a patient-derived breast tumor tissue sample can be used to detect the sensitivity of the patient's tumor cells to different drugs.

[0071] I. Plating of primary breast cancer cells: Breast tumor cells (5#) were cultured using the culture medium of the present invention in section (5) of Example 1 by the method described in section (1) of Example 2, and the cells were seeded into a 384-well plate at a density of 3,000 to 5,000 cells per well, and the cells were subjected to overnight adhesion culture.

[0072] II. Drug gradient experiments: (1) Prepare a drug storage plate by gradient dilution: 10 μL of the drug stock solution to be tested (the concentration of the drug stock solution is the maximum plasma concentration of the drug in the human body, C maxEach of the 100 μL of DMSO was taken and added to a 0.5 mL EP tube containing 20 μL of DMSO, and then 10 μL was aspirated from the EP tube and placed in a second 0.5 mL EP tube containing 20 μL of DMSO, i.e., the drug was diluted 1:3. The above process was repeated to gradually dilute until 8 or 9 concentrations required for dosing were obtained. Different concentrations of the drug were added to a 384-well drug storage plate. As a control, an equal volume of DMSO was added to each well of the solvent control group. In this example, the drugs to be tested are clinically approved antitumor drugs docetaxel (MCE), palbociclib (MCE), dovitinib (MCE), capecitabine (MCE), crizotinib (MCE) and etoposide (MCE).

[0073] (2) Using a high-throughput automated workstation (purchased from Perkin Elmer), different concentrations of drugs in a 384-well drug reservoir plate and solvent controls were added to a 384-well cell culture plate plated with breast tumor cells. Both drug and solvent control groups were composed of three replicate wells. The volume of drug added to each well was 100 nL.

[0074] (3) Cell activity detection: 72 hours after drug administration, the chemiluminescence value of the cultured cells after drug administration was detected using a Cell Titer-Glo assay kit (manufactured by Promega). The magnitude of the chemiluminescence value reflects the cell viability and the effect of the drug on the cell viability. The prepared Cell Titer-Glo detection solution was added to each well, and after mixing, the chemiluminescence value was detected using a microplate reader.

[0075] Use GraphPad Prism 7.0 software to generate graphs and calculate median inhibitory concentrations (IC) 50 was calculated.

[0076] (4) The results of drug susceptibility tests are shown in Figures 6A to 6F. Figures 6A-6F show the drug sensitivity of cultured breast tumor cells (5#) derived from surgically resected cancer tissue samples from breast tumor patients to three targeted drugs, palbociclib, crizotinib, and dovitinib, and three chemotherapy drugs, docetaxel, capecitabine, and etoposide. The results show that cells from the same patient have different sensitivities to different drugs.

[0077] Here, breast cancer cells (5#) were highly sensitive to docetaxel and etoposide, but less sensitive to capecitabine, crizotinib, dovitinib and palbociclib, suggesting that docetaxel and etoposide may be potential effective drugs for the treatment of breast tumors in patient 5#.

[0078] The results of this example demonstrate the potential application of culturing patient-derived breast cancer cells using the culture medium for mammary epithelial cells of the present invention and performing in vitro drug sensitivity testing for clinical drug screening and prediction of efficacy for patients with breast tumors. [Industrial Applicability]

[0079] The present invention provides a culture medium and a culture method for mammary epithelial cells. The cultured cells can be used for drug efficacy evaluation and screening. Therefore, the present invention is suitable for industrial application.

[0080] Although the present invention has been described in detail above with a general description and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made to the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention will fall within the scope of protection claimed by the present invention.

Claims

1. A culture medium for culturing mammary epithelial cells, comprising: A culture medium comprising β-estradiol, insulin-like growth factor 1, basic fibroblast growth factor, tumor necrosis factor-α, and optionally fibroblast growth factor 10.

2. Regarding the amount of each component in the culture medium, the following conditions were met: The concentration of β-estradiol is 5 nM to 50 nM; the concentration of insulin-like growth factor 1 is 10 ng / ml to 200 ng / ml; the concentration of basic fibroblast growth factor is 10 ng / ml to 200 ng / ml; a tumor necrosis factor-α concentration of 2 ng / ml to 100 ng / ml; The culture medium according to claim 1 , characterized in that it satisfies any one or more or all of the above requirements.

3. Regarding the amount of each component in the culture medium, the following conditions were met: β-estradiol concentration is 5 nM to 10 nM; the concentration of insulin-like growth factor 1 is between 20 ng / ml and 100 ng / ml; the concentration of basic fibroblast growth factor is 20 ng / ml to 100 ng / ml; a tumor necrosis factor-α concentration of 5 ng / ml to 50 ng / ml; Optionally added fibroblast growth factor 10 at a concentration of 0 ng / ml to 50 ng / ml; The culture medium according to claim 1 or 2, characterized in that it satisfies any one or more or all of the above requirements.

4. The culture medium according to any one of claims 1 to 3, further comprising amphiregulin, epidermal growth factor, insulin, B27, Y27632, neuregulin 1, fibroblast growth factor 7, A8301, and GlutaMAX-I.

5. Regarding the amount of each component in the culture medium, the following conditions were met: the amount of amphiregulin is 10ng / ml to 100ng / ml, the amount of epidermal growth factor is 2.5ng / ml to 20ng / ml, the amount of insulin is 1μg / ml to 10μg / ml, B27 is added at a volume ratio of 1:25 to 1:100, the amount of Y27632 is 5μM to 15μM, the amount of neuregulin 1 is 5nM to 20nM, the amount of fibroblast growth factor 7 is 2.5ng / ml to 20ng / ml, the amount of A8301 is 100nM to 500nM, and GlutaMAX-I is added at a volume ratio of 1:50 to 1:200, The culture medium according to claim 4, characterized in that it satisfies any one or more or all of the above requirements.

6. The culture medium according to any one of claims 1 to 5, further comprising an initial culture medium selected from the group consisting of DMEM / F12, DMEM, F12 or RPMI-1640, and one or more antibiotics selected from the group consisting of streptomycin / penicillin, amphotericin B and Primocin.

7. A culture method for culturing mammary epithelial cells, comprising a step of culturing primary mammary epithelial cells using the culture medium according to any one of claims 1 to 6.

8. The following steps: (1) preparing a culture medium according to any one of claims 1 to 6; (2) coating the culture vessel with an extracellular matrix gel; (3) inoculating the coated culture vessel with primary mammary epithelial cells and culturing them using the culture medium prepared in step (1); The culture method according to claim 7, comprising:

9. 1. A method for evaluating or screening a drug for treating a breast disease, comprising the steps of: (1) obtaining primary mammary epithelial cells and culturing them using the culture method according to claim 7 or 8; (2) selecting a drug to be tested and preparing different concentration gradients; (3) adding the drug at different concentrations prepared in step (2) to the mammary epithelial cells cultured in step (1); (4) detecting cell viability; and A method comprising:

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