Culture medium and culture 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 to a culture method for culturing such cells or organoids containing such cells. The present invention also relates to the use of cell progeny or organoids cultured by the culture medium and culture method of the present invention for 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 development and progression of breast tumors. However, many challenges remain in achieving sustainable culture of human mammary epithelial cells. Currently, two regenerative technologies have been reported that can be used for in vitro culture of human mammary epithelial cells. One is conditional reprogramming technology developed at Georgetown University in the United States, and the other is organoid technology developed at the Royal Netherlands Academy of Arts and Sciences. However, there are certain problems with the practical application of these two technologies: conditional reprogramming technology requires co-culturing the patient's own tumor cells with mouse-derived feeder cells, which may interfere with the analysis results of the tumor cells. The culture medium used in organoid technology has stem cell niche factors as its core component, but this culture technology requires a large amount of niche factors, is expensive, and has long culture cycles, making it difficult to operate. Consequently, neither of these technologies has actually seen large-scale advancement in the fields of basic research and new drug development.

[0003] In Patent Document 1, the present inventors describe a culture medium and culture method for in vitro culture of mammary epithelial cells without the use of feeder cells, at a manageable cost and with convenient operation. This is the first two-dimensional culture system for mammary epithelial stem cells in vitro that does not require feeder cells or Wnt agonists such as Wnt proteins and R-spondin family proteins 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 [Overview of the project]

[0005] Based on the original invention, it was unexpectedly discovered that applying additives such as tumor necrosis factor to a culture of mammary epithelial cells could achieve a more rapid promotion of continuous proliferation of mammary epithelial cells. 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 embodiments of the present invention, the amounts of each component in the culture medium of the present invention are as follows: 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. The tumor necrosis factor-α concentration is 2 ng / ml to 100 ng / ml, more preferably 5 ng / ml to 50 ng / ml, and, The concentration of fibroblast growth factor 10 added at will be between 0 ng / ml and 50 ng / ml. One or more of the following conditions must be met, or all of them must be met.

[0007] In embodiments of the present invention, the culture medium of the present 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] In a preferred embodiment, the amount of amphireglin 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 in 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 in a volume ratio of 1:50 to 1:200.

[0009] In embodiments of the present 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 preferred embodiments, when streptomycin / penicillin is used as the antibiotic(s), the concentration range for streptomycin is 25 μg / mL to 400 μg / mL, and the concentration range for penicillin is 25 U / mL to 400 U / mL. When amphotericin B is used as the antibiotic, the concentration range is 0.25 μg / mL to 4 μg / mL. When primocin is used as the antibiotic, the concentration range is 25 μg / mL to 400 μg / mL.

[0011] A second aspect of the present invention provides a method for culturing mammary epithelial cells, comprising the following steps: (1) preparing a culture medium for 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 them using the culture medium for 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, such as commercially available Matrigel (Corning: 354230) or BME (Trevigen: 3533-010-02). More specifically, the extracellular matrix gel is diluted in serum-free culture medium, which may be the initial culture medium in the present invention, such as 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 involves adding the diluted extracellular matrix gel to the culture vessel so that the bottom of the culture vessel is completely covered, and leaving it for longer than 30 minutes, preferably at 37°C, preferably 30 to 60 minutes, to coat the vessel. After the coating is complete, any excess extracellular matrix gel dilution is aspirated and discarded, and the culture vessel is ready for use.

[0013] Human mammary epithelial cells may be breast cancer tumor cells, normal mammary epithelial cells, or mammary epithelial stem cells. For example, the above tissue sample should be collected within 30 minutes of surgical excision or biopsy from the patient. More specifically, a tissue sample from a non-necrotic area should be taken in a sterile environment, approximately 0.5 cm². 3 Cut out the above volume, place it in pre-cooled antibiotic-containing DMEM / F12 culture medium, and transport it to the laboratory under ice cooling. For example, DMEM / F12 culture medium containing 50 U / mL to 200 U / mL (e.g., 100 U / mL) of penicillin and 50 μg / mL to 200 μg / mL (e.g., 100 μg / mL) of streptomycin is used for ice-cooled transport.

[0014] In a biosafety cabinet, the tissue sample is transferred to a cell culture dish and then rinsed with transport fluid. This washes away blood cells from the surface of the tissue sample and removes any unnecessary tissues such as skin and fascia from the surface of the tissue sample.

[0015] Transfer the rinsed tissue sample to a separate new culture dish and add 5 mL to 25 mL of transport solution. Using a sterile scalpel blade and forceps, cut the tissue sample to a diameter of 1 mm. 3 Divide the tissue into sections smaller than 100.

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

[0017] Next, centrifuge the cell suspension at at least 300 g / min for 3 to 10 minutes using a centrifuge. Discard the supernatant, resuspend the pellet in the culture medium of the present invention, and then, for culturing, inoculate the coated culture vessel at a density of 1×10 4 cells to 1×10 6 cells per well.

[0018] The third aspect of the present invention is a method for evaluating or screening a drug for treating breast diseases, which includes obtaining expanded progeny mammary epithelial cells using the culture medium and culture method of the present invention, and applying the obtained cells to the evaluation and screening of the effectiveness of the drug, particularly the in vitro effectiveness evaluation and screening of anti-tumor drugs.

[0019] Preferably, the method for evaluating or screening a drug for treating breast diseases includes the following steps: (1) The step of obtaining primary mammary epithelial cells and culturing them using the culture method of the present invention; (2) The step of selecting the drug to be tested and preparing it in different concentration gradients; (3) Adding the drugs with different concentrations prepared in step (2) to the mammary epithelial cells cultured in step (1); (4) Detecting the cell viability; It includes.

[0020] The beneficial effects of the present invention are as follows: (1) It realizes the rapid expansion of mammary epithelial cells in a short period, and a sufficient number of cells can be obtained within an effective time for basic research and drug screening applications; (2) The mammary epithelial cells obtained by in vitro culture using the culture medium and culture method of the present invention can maintain the heterogeneity in the patient from which the cells are derived and can be applied to the field of regenerative medicine; (3) The cultured mammary epithelial cells are not interfered 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, so it is not affected by the quality and amount of serum from different batches, and (5) In this technology, it is not necessary to add expensive stem cell niche factors such as R-spondin and Wnt family components to culture mammary epithelial cells. Also, 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, the cycle of expansion to obtain the same number of cells is shorter, and these are suitable for the application to the fields of drug efficacy evaluation, screening and toxicity testing, for example, high-throughput screening of new candidate compounds, and an in vitro high-throughput drug sensitivity function test for patients can be provided.

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

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

[0023] [Figure 1-1] Figures 1A-1F are graphs showing the effects of different concentrations of additive components in the culture medium on the proliferation of primary mammary epithelial cells in vitro. [Figure 1-2] Figure 1G is a graph showing the effects of different concentrations of additive components in the culture medium on the proliferation of primary mammary epithelial cells in vitro. [Figure 2] Figure 2A shows the results of fluorescence staining of the nuclei of primary mammary epithelial cells obtained by culturing primary mammary tumor cells isolated from clinical tissue samples of breast cancer up to the third passage using the culture medium for mammary epithelial cells of the present invention and the control culture medium in parallel, and then continuing the culture for 14 days (under a 100× microscope). Figure 2B shows the results of fluorescence labeling (DAPI) of the cell nuclei and statistical analysis of primary cells obtained by culturing primary cells from three different mammary tumor patients in parallel using the same culture method as in Figure 2A. "***" indicates P<0.001. [Figure 3] Figures 3A and 3B are comparative charts and curve graphs showing the effects of continuous culture using the culture medium and control culture medium of the present invention for a case of primary mammary epithelial cells, respectively. [Figure 4] Figures 4A and 4B are comparative charts and culture-day-population-doubling curve graphs, respectively, showing the effects of continuous culture using the culture medium and control culture medium of the present invention for another example of primary mammary epithelial cells. [Figure 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 fluorescence signal overlap imaging (MERGE) of mammary epithelial cells obtained by culturing primary breast cancer cells isolated from clinical tissue samples of breast cancer up to the third passage using the culture medium for mammary epithelial cells of the present invention and a control culture medium in parallel, and then continuing the culture for 14 days (under a 100× microscope). [Figure 6] Figures 6A-6F show dose-response curves of various drugs on mammary tumor cells cultured using the culture medium for mammary epithelial cells of the present invention. [Modes for carrying out the invention]

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

[0025] (1) Isolation of primary human mammary epithelial cells For sample transport and washing, a commercially available penicillin-streptomycin biantibody 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 solution will hereinafter be referred to as the "transport solution."

[0026] Breast tumor tissue samples 1#, 2#, 3#, 4#, and 5# were derived from cancerous tissue samples surgically removed from five breast tumor patients who were informed and gave their consent. One of these samples (1#) is used for the following explanation. The above tissue sample was collected within 30 minutes after surgical excision from the patient. More specifically, under sterile conditions, a volume of 0.5 cm³ was collected from a non-necrotic area. 3 The above tissue samples were cut, placed in a pre-cooled transport solution, and transported to the laboratory under ice-cold conditions.

[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 fluid. Blood cells on the surface of the tissue sample were washed away. Undesirable tissues such as skin and fascia were removed from the surface of the tissue sample.

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

[0029] Tissue sample sections were transferred to 50 mL centrifuge tubes and centrifuged at 1200 rpm for 5 minutes using a benchtop centrifuge. After carefully removing the supernatant from the centrifuge tubes using a pipette, 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). The mixture was then shaken and digested in a 37°C incubator for 30 to 90 minutes, and then centrifuged at 350 g / min for 5 minutes using a benchtop 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] Next, the cell suspension was centrifuged at 350 g / min for 5 minutes. After discarding the supernatant, the pellet was resuspended in the following basic culture medium.

[0031] Four other breast tumor tissue samples were isolated using the same process as described above.

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

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

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

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

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

[0037] [Table 1]

[0038] Here, "+" indicates that the culture medium with additives (sometimes multiple) promotes the proliferation of at least three primary breast cancer cells isolated from breast tumor tissue, compared to the basic medium. The number of "+"s indicates the degree of proliferation promotion. "-" indicates that the culture medium with additives (sometimes multiple) inhibits the proliferation of at least two primary breast cancer cells isolated from breast tumor tissue. "○" indicates that the culture medium with additives (sometimes multiple) has no significant effect on the proliferation of at least three 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 primary mammary gland tumor cells isolated from mammary gland tumor tissue in at least three cases. Among these additives, basic fibroblast growth factor and tumor necrosis factor α showed more potent effects. When all of the above factors were added to the basic culture medium, the degree of growth promotion was significantly increased.

[0040] (4) Optimization of the concentration of additives in the culture medium for human mammary epithelial cells The following seven different culture medium formulations were further prepared: Formula 1: Basic medium to which MEM-NEAA diluted 1:100 times, 1 mM sodium pyruvate, 50 ng / ml IGF1, 20 ng / ml bFGF, 10 ng / ml TNF-α, and 20 ng / ml FGF10 have been added. Composition 2: Basic 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. Composition 3: Basic medium to which 10 nM β-estradiol, 1:100-fold diluted MEM-NEAA, 50 ng / ml IGF1, 20 ng / ml bFGF, and 10 ng / ml TNF-α and 20 ng / ml FGF10 have been added. Composition 4: Basic medium to which 10 nM β-estradiol, 1:100-fold diluted MEM-NEAA, 1 mM sodium pyruvate, 20 ng / ml bFGF, and 10 ng / ml TNF-α and 20 ng / ml FGF10 have been added. Composition 5: Basic medium to which 10 nM β-estradiol, 1:100-fold diluted MEM-NEAA, 1 mM sodium pyruvate, 50 ng / ml IGF-1, and 10 ng / ml TNF-α and 20 ng / ml FGF-10 have been added. Composition 6: Basic medium to which 10 nM β-estradiol, 1:100-fold diluted MEM-NEAA, 1 mM sodium pyruvate, 50 ng / ml IGF1, 20 ng / ml bFGF, and 20 ng / ml FGF10 have been added. Composition 7: Basic medium supplemented with 10 nM β-estradiol, 1:100-fold diluted MEM-NEAA, 1 mM sodium pyruvate, 50 ng / ml IGF1, 20 ng / ml bFGF, and 10 ng / ml TNF-α.

[0041] Subsequently, 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. Each culture medium was added to a Matrigel-coated 96-well plate at a volume of 100 μl / well. Three replicate experiments were performed for each concentration. Primary breast cancer cells (2#) isolated from the mammary tumor tissue of Example (1) were seeded at a density of 500 cells per well in a Matrigel-coated 96-well culture plate and cultured at 37°C and 5% CO2 in formulation 1 containing different concentrations of β-estradiol. After the start of culture, the culture medium was changed every 3 days. Cells were counted on day 14 of culture. Formulation 1 was used as the experimental control; i.e., the β-estradiol content of formulation 1 was 0 nM. The results are shown in Figure 1A.

[0042] Culture media for human mammary epithelial cells containing non-essential amino acids (MEM-NEAA) at different concentrations were prepared by adding a non-essential amino acid (MEM-NEAA) additive (Thermo Fisher) to formulation 2 at dilution ratios of 1:200, 1:100, 1:50, and 1:20. Each culture medium formulation was added to a Matrigel-coated 96-well plate at a volume of 100 μl / well. Three replicate experiments were performed for each concentration. Primary cells were cultured and counted in the same manner as described above. Formulation 2 was used as the control, i.e., formulation 2 contained 0 non-essential amino acids. 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. Each culture medium formulation was added to a Matrigel-coated 96-well plate at a volume of 100 μl / well. Three replicate experiments were performed for each concentration. Primary cells were cultured and counted in the same manner as described above. Formulation 3 was used as the control, i.e., formulation 3 contained 0 sodium pyruvate. The results are shown in Figure 1C.

[0044] Next, 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 10 ng / ml, 20 ng / ml, 50 ng / ml, 100 ng / ml, and 200 ng / ml. Culture media containing different concentrations of IGF1 were added to Matrigel-coated 96-well plates at a volume of 100 μl / well. Three replicate experiments were performed for each concentration. Primary cells were cultured and counted in the same manner as described above. Formulation 4 was used as the experimental control, i.e., formulation 4 contained 0 IGF1. The results are shown in Figure 1D.

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

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

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

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

[0049] (5) Tests on the proliferation-promoting effect of culture media for human mammary epithelial cells Two different culture media formulations were further prepared as follows: 1. Control culture medium: The preparation procedure was as described in Patent Document 1. Specifically, 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) + Penicillin-streptomycin (Thermo Fisher SCIENTIFIC) at a volume ratio of 1:100 + 20 ng / ml human amphiregulin (R&D Systems) + 10 ng / ml EGF (Peprotech) + B27 (Thermo Fisher SCIENTIFIC) at a volume ratio of 1:50 + 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 "the 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 basic medium.

[0050] The proliferative effects of a 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 replicate experiments were performed per group.

[0051] Following the method in section (1) of Example 1, breast cancer cells (#1~#3) were isolated from breast tumor tissue and placed in a 24-well culture plate coated with Matrigel, with 3 × 10 cells per well. 4Cells were inoculated at a density of individual cells, two different culture medium formulations were used, and the cells were cultured at 37 °C and 5% CO2. The cells were subcultured at an equal inoculation density up to the third passage, and then cultured until the 14th day. Breast cancer cells cultured using different culture medium formulations were subjected to nuclear fluorescence staining and counted. Specifically, the cells in each group were rinsed twice with PBS buffer, fixed with 4% paraformaldehyde for 15 minutes, and then incubated at room temperature for 1 hour 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)), and then rinsed three times with TBST buffer for 3 minutes each. The TBST solution was removed. The cells were incubated with 1 μg / mL DAPI dye (Sigma) for 10 minutes. The cells were rinsed once with PBS. Under a 100× fluorescence microscope, after enclosing the cover glass with a drop of mounting medium (Thermo Fisher Scientific), random field photos were taken.

[0052] Representative results are shown in FIGS. 2A and 2B. FIG. 2A is a microscopic photograph after continuously culturing 2# breast cancer cells up to the third passage using the control culture medium and the culture medium of the present invention, and then culturing for 14 days at a density of 3×10 4 cells per well. FIG. 2B shows the statistical values of the DAPI counting results for breast cancer cells (1# - 3#) derived from different patients using the same procedure as 2#. The vertical axis is the statistical value of the counting results for DAPI staining under random fields. As shown in FIGS. 2A and 2B, compared with the control culture medium, the number of cells cultured up to the third passage using the culture medium of the present invention can increase by about 5.2 times 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 Primary breast cancer cells (6#, 7#) were isolated from the cancerous tissue of two breast tumor patients using the same method as in section (1) of Example 1. The isolated breast cancer cells were then counted using a hemocytometer, and the 6# cells were then placed at the same density (2 × 10 cells per well) into two 6-well plates coated with Matrigel (Corning). 5 The cells were inoculated in parallel. The coating method was as follows: Matrigel was diluted in a 1:50 ratio with DMEM / F12 culture medium to prepare an extracellular matrix diluent. 1.5 ml / well of the extracellular matrix gel diluent was added to a 6-well culture plate so that the bottom of the culture plate wells was completely covered. After standing in a 37°C incubator for 1 hour, the extracellular matrix gel diluent was removed to obtain a culture plate coated with extracellular matrix.

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

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

[0056] (2) Comparison of the effects of serial in vitro culture using different culture media for human mammary epithelial cells Human breast cancer cells in a culture plate were grown until they covered approximately 80% of the bottom surface area, at which point 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 centrifugation-treated cell pellet 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. Cells from each group were seeded in a separate 12-well culture plate coated with Matrigel at a subculturing ratio of 1:10, and culture was continued.

[0057] After subculturing, the cells were allowed to grow again until they covered approximately 80% of the bottom surface area of ​​the culture plate. The cultured cells were then digested again using the procedure described above, collected, and counted. The cells were then inoculated again in a 1:10 ratio and cultured continuously.

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

[0059] Figure 3A shows cell images taken under a 100× phase-contrast microscope of 6# cells cultured up to the second and fifth passages using the control culture medium and the culture medium of the present invention, respectively.

[0060] Figure 3B shows the continuous growth curves of 6# cells under the culture conditions of the culture medium and control culture medium of the present invention, drawn using GraphPad Prism 7.0 software. The horizontal axis represents the number of culture days, and the vertical axis represents the cell population doubling number.

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

[0062] Figure 4A shows cell images of cell #7 cultured up to the second and fourth passages after continuous culture following the same procedure using the control culture medium and the culture medium of the present invention, respectively. Figure 4B is a curve chart of culture days-cell population doubling number for cell #7 under the culture conditions of the culture medium of the present invention and the control culture medium, drawn using GraphPad Prism 7.0 software.

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

[0064] [Example 3] Identification of immune markers for mammary epithelial cells (1) The control culture medium and the culture medium of the present invention were prepared according to the description in section (5) of Example 1. (2) Cancer tissue the size of a soybean was obtained from a clinical surgical excision sample of a breast cancer patient, and primary breast cancer cells (3#) were isolated using the same method as in item (1) of Example 1. Primary breast cancer cells (3#) were cultured up to the third passage using the methods in items (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 (manufactured by 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 mammary gland tumors, while CK14 is recognized as an important biomarker for myoepithelial cells of mammary gland 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 the cells were incubated at 37°C for 15 minutes. The digested cells were then 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. The cells were collected, placed in a centrifuge tube, and centrifuged at 300 g / min for 5 minutes. After centrifugation, the cell pellets from each group were resuspended using the culture medium of the present invention and a known control culture medium, respectively. Cell counting was performed on the cell suspensions using a counting chamber. The cells were placed on Matrigel-coated coverslips, with 4 × 10 cells per coverslip. 4 The cells were inoculated at a density of [number] cells. The coating method was the same as in section (2) of Example 1. The cells of each group were cultured on coverslips using the culture medium of the present invention and a known control culture medium, respectively, to allow the cells to grow.

[0067] After 14 days of culture, the cells were rinsed twice with PBS buffer, fixed with 4% paraformaldehyde for 15 minutes, then incubated at room temperature for 1 hour with TBST (TBS + 0.1% Tween20) containing 1% BSA (Sangon Biotech (Shanghai)) and 1% Triton X-100, and rinsed three times with TBST buffer for 3 minutes each time. After removing the TBST solution, 50 μL of primary antibody dilution (CK8 antibody 1:100-fold dilution; CK14 antibody 1:1000-fold dilution) was added to the slide and incubated at 4°C for 12-16 hours, then rinsed three times with PBS for 3 minutes each time. The secondary antibody, anti-mouse IgG (H+L), F(ab')2 fragment (Alexa Fluor® 488 Conjugate) (8 μg / ml), was added and incubated at room temperature for 60 minutes, then rinsed three times with PBS for 3 minutes each time. Subsequently, the secondary antibody anti-rabbit IgG(H+L), F(ab')2 fragment (Alexa Fluor® 594 Conjugate) (8 μg / ml) was added, and the cells were incubated at room temperature for 60 minutes, rinsing three times with PBS for 3 minutes each time. The cells were incubated with 1 μg / mL of DAPI dye (Sigma) for 10 minutes, and rinsed once with PBS. After mounting the coverslip with one drop of mounting medium (Thermo Fisher Scientific), images were taken under a fluorescence microscope at 100× magnification.

[0068] The results are shown in Figure 5, which displays the results of immunofluorescence staining using a biomarker specific to luminal epithelial cells (CK8) and a biomarker specific to myoepithelial cells (CK14). Furthermore, the location and number of cell nuclei are indicated using DAPI fluorescence labeling, and imaging results generated by overlapping the three fluorescently labeled CK8, CK14, and DAPI using MERGE are also shown.

[0069] As shown in Figure 5, 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. On the other hand, when primary breast cancer cells derived from the same sample are cultured using a known control culture medium, only a small number of breast cancer cells are obtained. This demonstrates that the culture technique of the present invention can efficiently culture breast cancer cells.

[0070] [Example 4] In vitro drug sensitivity testing of breast cancer cells The following demonstrates, using surgically excised tissue samples from breast tumor patients as an example, that breast cancer cells cultured from patient-derived breast tumor tissue samples can be used to detect the sensitivity of a patient's tumor cells to different drugs.

[0071] I. Plating of primary breast cancer cells: Using the culture medium of the present invention as described in section (5) of Example 1, breast tumor cells (5#) were cultured according to the method described in section (1) of Example 2. The cells were inoculated into a 384-well plate at a density of 3000 to 5000 cells per well, and the cells were subjected to overnight adherent culture.

[0072] II. Drug Gradient Experiment: (1) A drug storage plate was prepared by concentration gradient dilution: 10 μL of drug stock solution to be tested (the concentration of the drug stock solution was the maximum plasma concentration of the drug in the human body C). maxEach of the two (prepared to twice the original concentration) was taken and added to a 0.5 mL EP tube containing 20 μL of DMSO. Then, 10 μL was aspirated from the EP tube and placed into 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 until the drug was gradually diluted to the concentration of 8 or 9 required for dosing. The drugs of different concentrations 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 the 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 drug and solvent control from a 384-well drug storage plate were added to a 384-well cell culture plate plated with mammary gland tumor cells. Both the drug group and the solvent control group were configured using three repeatable 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 cultured cells after drug administration was detected using the Cell Titer-Glo assay kit (manufactured by Promega). The magnitude of the chemiluminescence value reflects cell viability and the effect of the drug on 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] Using GraphPad Prism 7.0 software, create graphs and determine the median IC of inhibitory concentrations. 50 The result was calculated.

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

[0077] In this study, breast cancer cells (5#) were highly sensitive to docetaxel and etoposide, but less sensitive to capecitabine, crizotinib, dovitinib, and palbociclib. This suggests that docetaxel and etoposide may be potentially effective drugs for treating 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 of performing in vitro drug sensitivity testing for screening clinical drugs and predicting their efficacy in patients with mammary tumors. [Industrial applicability]

[0079] This 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, this invention is suitable for industrial applications.

[0080] Although the present invention has been described in detail above, along with the general description and specific embodiments, it will be apparent to those skilled in the art that some modifications or improvements can be made to the present invention. Accordingly, such 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, It contains β-estradiol, insulin-like growth factor 1, basic fibroblast growth factor, tumor necrosis factor-α, amphiregulin, epidermal growth factor, insulin, B27, Y27632, neuregulin 1, fibroblast growth factor 7, A8301, and GlutaMAX-I. Regarding the amounts of each component in the culture medium, the following conditions apply: The concentration of β-estradiol is 5 nM to 50 nM. The concentration of insulin-like growth factor 1 is between 10 ng / ml and 200 ng / ml. The concentration of basic fibroblast growth factor should be between 10 ng / ml and 200 ng / ml. The tumor necrosis factor-α concentration is between 2 ng / ml and 100 ng / ml. The amount of amphireglin is 10 ng / ml to 100 ng / ml. The amount of epidermal growth factor should be between 2.5 ng / ml and 20 ng / ml. The insulin dose is between 1 μg / ml and 10 μg / ml. The volume ratio of B27 to the culture medium (B27: culture medium) is 1:25 to 1:

100. The amount of Y27632 is between 5 μM and 15 μM. The amount of Neuregulin 1 is between 5 nM and 20 nM. The amount of fibroblast growth factor 7 is between 2.5 ng / ml and 20 ng / ml. The amount of A8301 is between 100 nM and 500 nM. The volume ratio of GlutaMAX-I to the culture medium (GlutaMAX-I: culture medium) is 1:50 to 1:

200. A culture medium characterized by satisfying all of the following conditions.

2. Regarding the amounts of each component in the culture medium, the following conditions apply: The concentration of β-estradiol 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 between 20 ng / ml and 100 ng / ml. The tumor necrosis factor-α concentration is between 5 ng / ml and 50 ng / ml. The culture medium according to claim 1, characterized in that it satisfies one or more or all of the following conditions.

3. It further contains fibroblast growth factor 10, The culture medium according to claim 1 or 2, characterized in that the concentration of fibroblast growth factor 10 in the culture medium is 50 ng / ml or less.

4. The culture medium according to any one of claims 1 to 3, 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.

5. A method for culturing mammary epithelial cells, characterized by comprising the step of culturing primary mammary epithelial cells using a culture medium described in any one of claims 1 to 4.

6. The following steps: (1) A step of preparing a culture medium according to any one of claims 1 to 4, (2) A step of coating the culture vessel with an extracellular matrix gel, (3) The process of inoculating primary mammary epithelial cells into a coated culture vessel and culturing them using the culture medium prepared in step (1), The culture method according to claim 5, characterized by including the following:

7. A method for evaluating or screening drugs for the treatment of breast disease, comprising the following steps: (1) A step of obtaining primary mammary epithelial cells and culturing them using the culture method described in claim 5 or 6, (2) A step of selecting the drug to be tested and preparing different concentration gradients, (3) A step of adding drugs of different concentrations prepared in step (2) to mammary epithelial cells cultured in step (1), (4) A step to detect the cell viability, A method characterized by including

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