WO2021088119A1 - 原代乳腺上皮细胞培养基、培养方法及其应用 - Google Patents

原代乳腺上皮细胞培养基、培养方法及其应用 Download PDF

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WO2021088119A1
WO2021088119A1 PCT/CN2019/119116 CN2019119116W WO2021088119A1 WO 2021088119 A1 WO2021088119 A1 WO 2021088119A1 CN 2019119116 W CN2019119116 W CN 2019119116W WO 2021088119 A1 WO2021088119 A1 WO 2021088119A1
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cells
primary
breast cancer
culture medium
cell
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PCT/CN2019/119116
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French (fr)
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刘青松
刘飞扬
梅沪生
王文超
赵明
蒋宗儒
任涛
王黎
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合肥中科普瑞昇生物医药科技有限公司
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Priority to US17/775,106 priority Critical patent/US20220389379A1/en
Priority to EP19951810.1A priority patent/EP4056685A4/en
Priority to JP2022526407A priority patent/JP7373872B2/ja
Publication of WO2021088119A1 publication Critical patent/WO2021088119A1/zh

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Definitions

  • the present invention belongs to the field of medical technology. Specifically, it relates to a culture medium and a culture method for culturing or amplifying primary breast epithelial cells in vitro, and also relates to the application of the cultured cells in the efficacy evaluation and screening of drugs.
  • Breast cancer is one of the most important malignant tumors that affect women's health. The latest statistics show that on a global scale, the incidence and mortality of breast cancer rank first and second respectively in the incidence and mortality of female malignant tumors. In recent years, although people have made a lot of progress in the molecular typing and pathogenesis of breast cancer, the current standard drug treatment methods for breast cancer are still based on hormones and cytotoxic drugs, and there is a lack of personalized and precise medication guidance. . Because of the diversity and complexity of breast cancer classification and its high degree of heterogeneity, it is difficult to truly predict the efficacy of clinical medications based on molecular or genetic diagnosis without auxiliary functional testing (Adam A. Friedman et al., Nat Rev. Cancer, 15(12):747-56, 2015).
  • Functional testing refers to a method of testing the sensitivity of anti-tumor drugs on cancer patient cells in vitro.
  • the key to applying this method is to develop a tumor cell model that has a short growth cycle and can represent the biological characteristics of breast cancer patients.
  • the cell model should be easy to operate to quickly and efficiently predict the curative effect of clinical medication, so as to give cancer patients precise medication guidance in time.
  • the success rate of establishing cell models in vitro with primary tumor cells taken from cancer patients is often very low, the growth cycle is long, and there are problems such as excessive proliferation of interstitial cells such as fibroblasts, which restrict the development of this field.
  • two techniques for culturing primary epithelial cells/stem cells are relatively mature in the application field of tumor cell functional testing.
  • Cell reprogramming technology is a technology that co-cultures patients’ autologous primary epithelial cells with murine-derived feeder cells.
  • the presence of these murine-derived cells can interfere with the patient The results of drug sensitivity test of autologous primary cells; but if the mouse-derived feeder cells are removed, the patient's autologous primary cells will be out of the reprogramming environment, and the cell proliferation rate and intracellular signaling pathways will be significantly changed (Liu et al., Am J Pathol, 183(6): 1862-1870, 2013; Liu et al., Cell Death Dis., 9(7): 750, 2018), which greatly affects the response of patients’ autologous primary cells to drugs .
  • Organoid technology is a technology that embeds the patient’s autologous primary epithelial cells in an extracellular matrix for three-dimensional culture in vitro. This technology does not require feeder cells, so there is no interference problem of mouse-derived feeder cells, but the culture of organoid technology A variety of specific growth factors need to be added in the base, which is expensive and not suitable for popularization in clinics for large-scale applications. In addition, organoids need to embed cells in extracellular matrigel during the entire culture process. The plating steps of cell seeding, passage and drug sensitivity test are more cumbersome and time-consuming than 2D culture operations. The size of organoids is not easy to control, and some organoids grow too large and cause internal necrosis.
  • organoid technology compared with 2D culture technology, the operability and applicability of organoid technology is not strong, and it requires professional and technical personnel to operate it. It is not suitable for large-scale and wide application in clinical in vitro drug sensitivity testing (Nick Barker, Nat Cell Biol, 18(3):246-54, 2016).
  • the cultured breast cancer tumor cells can represent the biological characteristics of breast cancer patients.
  • the present invention aims to address the deficiencies of the prior art and provide a primary mammary epithelial cell culture medium for culturing primary mammary epithelial cells and a method for culturing primary mammary epithelial cells using the medium.
  • the mammary substitute epithelial cell culture medium and culture method can achieve the purpose of short in vitro culture period, controllable cost, convenient operation and no interference from exogenous cells.
  • One aspect of the present invention is to provide a primary cell culture medium for culturing primary breast epithelial cells, which contains Amphiregulin.
  • the content of bimodulin is preferably 10 ng/ml or more, and from the viewpoint of cost, it is preferably 10 ng/ml to 100 ng/ml.
  • the primary cell culture medium of the present invention preferably further contains: epidermal growth factor (EGF), insulin (Insulin), B27, ROCK kinase inhibitor Y27632, neurotonin 1 (Neuregulin1), fibroblast growth factor 7 (FGF7), One or more or all of the TGF ⁇ type I receptor inhibitor A8301 and the P38/MAPK inhibitor SB202190.
  • EGF epidermal growth factor
  • Insulin insulin
  • B27 ROCK kinase inhibitor Y27632
  • neurotonin 1 Neurotonin 1
  • FGF7 fibroblast growth factor 7
  • TGF ⁇ type I receptor inhibitor A8301 and the P38/MAPK inhibitor SB202190 One or more or all of the TGF ⁇ type I receptor inhibitor A8301 and the P38/MAPK inhibitor SB202190.
  • the content of EGF is 2.5ng/ml-20ng/ml; the content of insulin is 1 ⁇ g/ml-10 ⁇ g/ml; the B27 is diluted with a final concentration of 1:25 to 1:100; the Y27632
  • the content is 5 ⁇ M ⁇ 15 ⁇ M; the content of the neurotonin 1 is 5nM ⁇ 20nM; the content of the FGF7 is 2.5ng/ml ⁇ 20ng/ml; the content of the A8301 is 100nM ⁇ 500nM; the content of the SB202190 is 100nM ⁇ 500nM.
  • the medium formula contains bimodulin, does not contain uncertain components such as serum, bovine pituitary extract, and does not contain Wnt agonists, R-spondin family proteins, BMP inhibitors and other niche factors necessary for organ culture, and do not contain fibroblast growth factor 10 (FGF10), Nicotinamide and N-Acetylcysteine ,
  • FGF10 fibroblast growth factor 10
  • Nicotinamide and N-Acetylcysteine
  • the primary breast epithelial cells may be breast cancer tumor cells, normal breast epithelial cells, and breast epithelial stem cells.
  • One aspect of the present invention is to provide a method for culturing primary breast epithelial cells, which includes the following steps:
  • the extracellular matrix glue uses a low growth factor extracellular matrix glue, for example, commercially available Matrigel (Corning: 354230) or BME (Trevigen: 3533-010-02) can be used. More specifically, the extracellular matrigel is diluted with a serum-free medium.
  • the medium may be the primary cell culture medium of the present invention or DMEM/F12 (Corning: R10-092-CV).
  • the dilution ratio of extracellular matrigel is 1:50-400, preferably 1:100-200.
  • the coating method is to add the diluted extracellular matrigel to the culture vessel to completely cover the bottom of the culture vessel, leave it to stand and coat for more than 30 minutes, preferably at 37°C for coating, preferably coating 30-60 minute. After the coating is finished, aspirate and discard the excess extracellular matrigel diluent, and incubate it for use.
  • Primary breast epithelial cells can be derived from breast cancer tissue samples and para-cancerous tissue samples, for example.
  • Breast cancer tissue samples are, for example, derived from surgically removed cancer tissue samples from breast tumor patients who have explained and obtained consent, and the adjacent tissue samples are collected from breast tissue that is at least 5 cm away from the breast cancer tissue. Collect the above-mentioned tissue samples within half an hour after the patient's surgical resection or biopsy. More specifically, in a sterile environment, cut tissue samples from non-necrotic sites with a volume above 0.5 cm 3 and place them in a pre-cooled 10-50 mL DMEM/F12 medium. The medium is contained in a plastic non-necrotic medium.
  • DMEM/F12 medium contains 50-200U/mL (e.g. 100U/mL) penicillin and 50-200U/mL (e.g. 100U/mL) streptomycin ( Hereinafter referred to as transport fluid).
  • tissue sample In the biological safety cabinet, transfer the tissue sample to a cell culture dish, rinse the tissue sample with transport fluid, wash off the blood cells on the surface of the tissue sample, and remove unnecessary tissues such as skin and fascia on the surface of the tissue sample.
  • tissue sample fragments to the centrifuge tube, centrifuge at least 1000 rpm for 3-10 minutes in a benchtop centrifuge; then carefully remove the supernatant from the centrifuge tube with a pipette, and then use 5-25mL collagenase II (0.5 -5mg/mL, for example, 1mg/mL) and collagenase IV (0.5-5mg/mL, for example, 1mg/mL) serum-free DMEM/F12 medium resuspended, placed on a 37°C constant temperature shaker for shaking digestion, the time is at least 1 hour (digestion time depends on the sample size; if the sample is larger than 1g, the digestion time is increased to 1.5-2 hours); after that, centrifuge at least 300g/min in a benchtop centrifuge for 3-10 minutes, discard the supernatant, and after digestion Resuspend the tissue cells in 5-25 mL of DMEM/F12 medium containing, for example, 10% calf serum, grind and sieve
  • the cell suspension is then centrifuged in a centrifuge at at least 300 g/min for 3-10 minutes, the supernatant is discarded, and then resuspended in the primary cell culture medium of the present invention.
  • the oxygen concentration can be normal oxygen (15-20% oxygen concentration) or hypoxia ( 0.5-4% oxygen concentration).
  • fresh primary cell culture medium is replaced every 4 days, and the digestion and passage are carried out when the primary breast epithelial cells grow to a cell density of about 80% to 90% of the bottom area of the culture flask.
  • This inoculation step does not need to use feeder cells, and compared with the cell condition reprogramming technology, the operation steps of culturing and irradiating feeder cells are eliminated. Compared with the organoid technique, this step does not require mixing the primary cells and Matrigel on ice to form glue droplets, and waiting for the glue droplets to solidify before adding the culture medium.
  • the pre-coated culture vessel can be used directly for the primary generation. Cell seeding, in addition, only a small amount of diluted extracellular matrix glue is needed to coat the culture vessel. Compared with organoid technology, it saves the amount of expensive extracellular matrix glue and simplifies the operation steps.
  • T25 cell culture flask Suspend the digested cells, centrifuge at at least 300g/min for 3-10 minutes, resuspend the digested single cells using the primary cell culture medium of the present invention, and place the resulting cell suspension outside the coated cells Continue to expand the culture in Matrigel T25 cell culture flask.
  • the coating operation of T25 cell culture flask is the same as step (2).
  • the expanded mammary epithelial cells grow in 2D, which avoids the uneven size of organoids and the internal necrosis of overgrown organoids that occur in the expansion of organoid technology.
  • breast epithelial cells cultured by the primary breast epithelial cell culture method of the present invention can be used for drug efficacy evaluation and screening, including the following steps:
  • primary breast epithelial cells and particularly preferably obtain cancer tissue samples or biopsy cancer tissue samples derived from breast cancer patients, isolate primary breast epithelial cells, culture and expand primary breast epithelial cells according to the method described above cells (particularly primary breast cancer tumor cells) of the order of at least 105, preferably at least 106 the number of cells of magnitude.
  • the drug takes its maximum plasma concentration Cmax as a reference, and uses 2-5 times Cmax as the initial concentration to dilute multiple different drug concentration gradients, such as 5-10, preferably 6-8 drug concentration gradients.
  • step (1) Digest the mammary gland epithelium cultured in step (1) into a single cell suspension, count it with a hemocytometer, and dilute the single cell suspension with the primary cell culture medium of the present invention containing extracellular matrix glue,
  • the diluted cell suspension is uniformly added to the multi-well plate at a density of 1000-10000 cells per well, for example, 50 ⁇ L of cell diluent per well, and adhered overnight.
  • This step avoids the problem of cell reprogramming technology that interferes with the primary cell count and subsequent primary cell viability detection due to the presence of feeder cells, and there is no need to combine the cell suspension and matrigel on ice like the organoid technology.
  • the cumbersome steps of mixing, embedding and re-laying greatly simplify the operation process and enhance the operability and practicability of the technology. Since the seeded cells are a single-cell suspension rather than a 3D structure like organoids, compared with the organoid technology, the number of cells plated is more uniform, and the number of cells between the wells produced by the plating is smaller, and it is more suitable for carrying out Subsequent high-throughput drug screening operations.
  • a high-throughput automated workstation is used to add gradient dilutions of the selected traditional chemotherapy drugs, targeted drugs, antibody drugs, or several drug combinations to the adherent cells obtained in step (4). deal with.
  • the Cell-Titer Glo Luminescence Cell Viability Detection Kit (Promega: G7573) is used to detect the survival rate of mammary epithelial cells for drug activity screening.
  • the phenomenon that feeder cells in the cell reprogramming technology interfere with the detection result will not occur. Since the cells grow in 2D, the action time with the drug is also shorter than the drug detection time of the organoid technology (the average administration time of the organoid technology is 6 days).
  • the cultured primary mammary epithelial cells are not interfered by fibroblasts, adipocytes and other interstitial cells, and purified mammary epithelial cells can be obtained;
  • the medium components do not contain serum, so it is not affected by the quality and quantity of different batches of serum;
  • the amplified mammary epithelial cells can also be serially passaged ;
  • the passage step does not require operation on ice and dissociation of Matrigel, and the digestion and passage of cells can be completed within 10-15 minutes;
  • the culture cost is controllable: the primary breast cancer medium does not need to add expensive Wnt agonists, R-spondin family proteins, BMP inhibitors, FGF10 and other factors. It is an organoid medium for existing primary breast epithelial cells The simplification and improvement of cell inoculation does not require the use of a higher concentration of extracellular matrix to mix with primary cells to form droplets, but only a small amount of diluent prepared by extracellular matrix glue, which saves the cost of extracellular matrix. Dosage
  • breast epithelial cells derived from humans or other mammals can be cultured, including breast cancer tumor cells, normal breast epithelial cells, breast epithelial stem cells, or containing at least any of these cells Organization.
  • organoids can also be formed from at least one of the cells and the tissues.
  • the cells obtained by the culture method of this embodiment can be used in regenerative medicine, basic medical research on breast epithelial cells, screening of drug responses, and development of new drugs derived from breast diseases.
  • Figure 1 is an inverted phase-contrast microscope photograph of primary breast epithelial cells obtained by culturing cells isolated from clinical breast tissue samples using the primary cell culture method of the present invention.
  • Figure 2 shows the primary breast cancer tumor cells isolated from two different breast cancer clinical tissue samples (HMFL-XN30, HMFL-XN22) were inoculated in cultures coated with extracellular matrix gel and without any coating. Microscopic photograph of cells cultured in the plate.
  • Figure 3 is used to illustrate the proliferation effect of bimodulin on primary breast cancer tumor cells.
  • Figure 4 is a comparison diagram of cell growth curves obtained by culturing cells obtained from two cases of breast cancer clinical tissue samples (HMFL-XN12, HMFL-XN21) using cell conditional reprogramming technology, the technology of the present invention, and organoid technology, and A photo of HMFL-XN21 under the microscope at the 27th day of culture.
  • Fig. 5 is a photograph of breast cancer tumor cells cultured to the 9th day and cultured to the 22nd day of cells isolated from a clinical tissue sample of breast cancer (HMFL-XN12) under an inverted microscope using the technology of the present invention.
  • Fig. 6 is a comparison of immunofluorescence staining results of breast cancer tumor cells cultured with the technique of the present invention on cells (HMFL-XN7) isolated from a sample of breast cancer surgically resected sample with the immunohistochemical results of the original tissue section of the tissue sample itself.
  • Figure 7 shows the genetic mutation consistency analysis of breast cancer tumor cells of different generations obtained by culturing the cells obtained from two cases of breast cancer surgical resection samples using the technique of the present invention, and the chromosomal karyotype analysis results of one of the cases.
  • Fig. 8 shows the tumor formation in mice of primary breast cancer tumor cells derived from the cancer tissues of two cases of pathologically diagnosed triple-negative breast cancer patients.
  • Fig. 9 shows the dose-effect curve of different chemotherapeutic drugs and targeted drugs of primary breast cancer tumor cells cultured using the technology of the present invention and the calculated half inhibition rate.
  • HMFL-XN1 Breast cancer tissue samples were derived from five cases of breast cancer patients who had explained and obtained consent for surgical resection of cancer tissue samples. They were HMFL-XN1, HMFL-XN3, HMFL-XN4, HMFL-XN6, and HMFL-XN8. Let's take one of the samples (HMFL-XN1) for illustration. Collect the above-mentioned tissue samples within half an hour after the patient's surgical resection or biopsy. More specifically, in a sterile environment, cut a tissue sample from a non-necrotic site with a volume of 0.5 cm 3 or more, and place it in a pre-cooled 20 mL DMEM/F12 medium (manufactured by Corning).
  • transport solution 100U/mL penicillin and 100U/mL streptomycin
  • tissue sample (HMFL-XN1) to a 100mm cell culture dish, rinse the tissue sample with transport fluid, wash off the blood cells on the surface of the tissue sample, and remove the skin, fascia, etc. on the surface of the tissue sample Unwanted organization.
  • tissue sample fragments to a 50mL centrifuge tube, centrifuge at 1200 rpm for 5 minutes in a benchtop centrifuge; then use a pipette to carefully remove the supernatant from the centrifuge tube, and then use 10mL collagenase II (1mg/mL) and Resuspend collagenase IV (1mg/mL) in serum-free DMEM/F12 medium, and place it on a constant temperature shaker at 37°C for shaking and digestion for 1 hour; then centrifuge at 350g/min for 5 minutes in a benchtop centrifuge and discard it.
  • the digested tissue cells are resuspended in 10mL DMEM/F12 medium containing 10% calf serum, ground and sieved, the cell sieve aperture is 100 ⁇ m, and the sieved cell suspension is collected in a 50mL centrifuge tube; Count on a hemocytometer.
  • the cell suspension was centrifuged in a centrifuge at 350 g/min for 5 minutes, the supernatant was discarded, and then resuspended in the primary cell culture medium of the present invention.
  • Matrigel (registered trademark) (manufactured by BD Biotech) was diluted 1:100 with serum-free DMEM/F12 medium to prepare an extracellular matrix diluent, and 200 ⁇ l/well of extracellular matrix was added to a 48-well culture plate Dilute the solution to completely cover the bottom of the plate well. Let stand for 1 hour in a 37°C incubator. After 1 hour, the extracellular matrix diluent was removed to obtain a culture plate coated with Matrigel.
  • Second configure the basal medium Add GlutaMAX-I (manufactured by Thermo Fisher SCIENTIFIC) to a commercially available DMEM/F-12 medium at the indicated concentration (1:100 dilution), and add human insulin (manufactured by Sigma) at a final concentration of 10 ⁇ g/ml , Add ROCK kinase inhibitor Y27632 (manufactured by Sigma) at a final concentration of 10 ⁇ M, and add penicillin-streptomycin (manufactured by Thermo Fisher Scientific) at a dilution ratio of 1:100 to prepare a basic medium.
  • GlutaMAX-I manufactured by Thermo Fisher SCIENTIFIC
  • human insulin manufactured by Sigma
  • ROCK kinase inhibitor Y27632 manufactured by Sigma
  • penicillin-streptomycin manufactured by Thermo Fisher Scientific
  • HMFL-XN1 Breast cancer tumor cells isolated from breast cancer tissue in step (1) of this example were seeded in a 48-well culture plate coated with Matrigel at a cell density of 1 ⁇ 10 4 cells/well. Under the conditions of °C, 5% CO 2 concentration, and 20% oxygen concentration, an equal number of freshly isolated breast cancer tumor cells (HMFL-XN1) were cultured under different medium formula conditions. The culture medium was replaced every 4 days after the start of the culture.
  • the medium added with the additive has the effect of promoting the proliferation of at least three primary breast cancer tumor epithelial cells isolated from breast cancer tissue;
  • “-” means adding the The medium of the additive has the effect of inhibiting the proliferation of at least two cases of primary breast cancer tumor epithelial cells isolated from breast cancer tissue;
  • “ ⁇ ” means that the medium with the additive has an effect on the original isolated from breast cancer tissue. The proliferation of at least three cases of breast cancer tumor epithelial cells had no obvious influence.
  • the breast epithelial cells derived from the cancer tissue and the breast epithelial cells derived from the adjacent tissue were separately isolated from the cancer tissue and the adjacent breast tissue of the same breast cancer patient.
  • the para-cancerous breast tissue samples were collected from breast tissue at least 5 cm away from the breast cancer tissue.
  • the breast cancer tumor cells derived from the cancer tissue were counted with a hemocytometer, and then inoculated into a 12-well plate coated with Matrigel (registered trademark) (manufactured by BD Biotech Co., Ltd.) at a density of 4 ⁇ 10 4 cells/well.
  • Matrigel registered trademark
  • Figure 1(A) is a microscopic photograph of breast cancer tumor cells (HMFL-XN11) isolated from a breast tumor sample resected by a clinical operation from the start of inoculation to the 7th day (under a 50-fold inverted phase contrast microscope). Observation under the microscope shows that the primary breast cancer tumor cells derived from the cultured cancer tissues are relatively pure and do not contain interstitial cells such as fibroblasts.
  • FIG. 1 (B) of Figure 1 is a primary normal breast epithelial cell (HMFL-XN11-N) isolated from a paracancerous tissue sample from a patient with the same source as HMFL-XN11, inoculated into Matrigel coating at a density of 4 ⁇ 10 4 cells/well The processed 12-well plate was cultured from the inoculation to the 7th day under the microscope (under a 50-fold inverted phase contrast microscope).
  • Figure 1 (A) and (B) illustrate that the primary breast epithelial cell culture medium and culture method of the present invention can achieve high efficiency on breast cancer tumor cells derived from breast cancer tissue and normal breast epithelial cells derived from adjacent tissues. In vitro culture. None of the cultured primary cells contained interstitial cells such as fibroblasts.
  • FIG. 1 shows that the primary cell culture medium and culture method of the present invention can be used to cultivate two different cell subpopulations that constitute the breast: luminal cells and myoepithelial cells, which achieves the maintenance of heterogeneous tumor samples. Sexual in vitro culture.
  • the primary breast cancer tumor cells (HMFL-XN30) derived from the breast cancer tissue were isolated from the cancer tissue of a breast cancer patient.
  • primary breast cancer tumor cells HMFL-XN30 of the same cell number (4 ⁇ 10 4 cells/well) were seeded on a 12-well plate coated with Matrigel (registered trademark) (manufactured by BD Biotech).
  • Matrigel registered trademark
  • the tumor cells are cultured.
  • FIGS. 1 and (F) of Figure 1 are cytoscopic comparisons of breast epithelial cells derived from cancer tissues cultured under normoxia and hypoxia conditions, respectively. From Figure 1 (E) and (F), it can be confirmed that the culture medium and culture method of the present invention can treat cancer both under normoxic conditions (20% oxygen concentration) or under hypoxic conditions (2% oxygen concentration). Primary breast epithelial cells derived from tissues are cultured efficiently.
  • step (1) of Example 1 The same method as step (1) of Example 1 was used to isolate primary breast cancer tumor cells (HMFL-XN30, HMFL-XN22) derived from the cancer tissue from the cancer tissues of two breast cancer patients.
  • an equal number (4 ⁇ 10 4 cells/well) of primary breast cancer tumor cells HMFL-XN30 were respectively inoculated into Matrigel (registered trademark) (manufactured by BD Biotechnology) coated 12-well plates and untreated 12-well plates. Any processed 12-well plate.
  • HMFL-XN22 (P3) is the third generation of breast cancer tumor cells that have been continuously cultured to the third generation after HMFL-XN22 is coated with Matrigel and without Matrigel. The rest of the steps are the same as those of HMFL-XN30.
  • Figure 2 is a microscopic photograph of breast cancer tissue-derived primary breast cancer tumor cells HMFL-XN30 and HMFL-XN22 (P3) cultured to the 10th day under Matrigel and uncoated conditions, respectively. According to Figure 2, it can be confirmed that the culture plate coated with Matrigel is more conducive to the proliferation of breast cancer tumor cells than the culture plate without any treatment.
  • step (1) of Example 2 The same method as in step (1) of Example 2 was used to prepare a culture medium for primary mammary gland epithelial cells.
  • the bimodulin was removed from the formula of the primary breast epithelial cell culture medium, and another primary culture medium without bimodulin was prepared.
  • Example 2 Using the same method as the step (1) of Example 1 to obtain two cases of breast cancer patients with primary breast cancer cells (HMFL-XN2, HMFL-XN12) derived from surgical resection of cancer tissue samples and one case of breast cancer Primary breast cancer tumor cells (HMFL-XN13) from the patient's puncture sample.
  • HMFL-XN2, HMFL-XN12 primary breast cancer cells
  • HMFL-XN13 breast cancer Primary breast cancer tumor cells
  • the primary breast cancer tumor cells (HMFL-XN2) derived from surgically removed cancer tissues were respectively inoculated into Matrigel (registered trademark) (manufactured by BD Biotech ) at the same seeding density (5 ⁇ 10 4 cells/hole) for coating treatment Inside the 12-well plate.
  • the primary mammary epithelial cell culture medium of the present invention containing bimodulin and the primary culture medium without bimodulin are used to culture the cells at 37° C. under the condition of an oxygen concentration of 20%, with two replicate holes in each group.
  • Figure 3(A) shows the cells (HMFL-XN2) isolated from the same breast cancer patient were inoculated according to the same cell number (5 ⁇ 10 4 cells/well). Microscopic pictures of tumor cell clones formed after culturing in the primary culture medium for 6 days (under a 100-fold inverted phase contrast microscope).
  • Figure 3(B) shows three cases of breast cancer tumor cells (HMFL-XN2, HMFL-XN-12 and HMFL-XN13) derived from breast cancer tissue using the primary culture medium without and containing biregulin, respectively The cell count results after 10 days of culture, two replicate wells per group. It can be seen from Figure 4 that with the cells cultured in the medium group without bimodulin as a control, the addition of bimodulin can promote the proliferation of primary mammary epithelial cells, and the addition of this component can increase by at least 50% and up to 250%. Promote the proliferation of primary breast epithelial cells.
  • step (1) of Example 2 The same method as in step (1) of Example 2 was used to prepare a culture medium for primary mammary gland epithelial cells.
  • a control another medium for cell conditional reprogramming technology (hereinafter also referred to as “cell conditional reprogramming technology medium”) was prepared.
  • the medium formula is shown in Table 2.
  • a culture medium for mammary gland organoids hereinafter also referred to as "organoid technology medium” was prepared.
  • organoid technology medium for mammary gland organoids
  • HMFL-XN12 and HMFL-XN21 Two cases of primary breast cancer tumor cells (HMFL-XN12 and HMFL-XN21) derived from breast cancer tissues were obtained using the same method as step (1) of Example 1. Then, HMFL-XN12 and HMFL-XN21 were cultured at the same density (4 ⁇ 10 4 cells/well) under the following three culture conditions:
  • the technology of the present invention the primary breast cancer tumor cells were inoculated into a 12-well plate coated with Matrigel (registered trademark) (manufactured by BD Biotechnology) at a seeding density of 4 ⁇ 10 4 cells/well, using 2 mL of Culture the primary breast epithelial cell culture medium of the present invention;
  • B. Cell conditional reprogramming technology inoculate primary breast cancer tumor cells at a seeding density of 4 ⁇ 10 4 cells/well to the mouse fibroblast cell line J2 cells (purchased from Kerafast) that have been irradiated by gamma rays Above, the cell conditional reprogramming technology medium was used for culture in a 12-well plate (for specific steps, see Liu et al., Am J Pathol, 183(6): 1862-1870, 2013);
  • C. Inoculate primary breast cancer tumor cells into a 12-well plate coated with Matrigel (registered trademark) (manufactured by BD Biotechnology) at a seeding density of 4 ⁇ 10 4 cells/well, and culture using 2 mL organoid technology The base is cultured in a 12-well plate.
  • Matrigel registered trademark
  • the cells cultured under the three culture conditions are exchanged every 4 days.
  • HMFL-XN12 and HMFL-XN21 For the primary breast cancer tumor cells (HMFL-XN12 and HMFL-XN21) cultured using the technology of the present invention, when they grow to about 80% of the bottom area of the culture plate, discard the culture in the original 12-well plate Based on the supernatant, add 1mL 0.05% trypsin (Thermo Fisher: 25300062) to digest the cells, incubate at 37°C for 15 minutes, 15 minutes later, use 10% (v/v) calf serum, 100U/mL penicillin and Resuspend the digested cells in 5 mL of 100U/mL streptomycin DMEM/F12 culture solution and collect them in a centrifuge tube, centrifuge at 350g/min for 5 minutes, use the culture medium of the present invention to resuspend the cell pellet after centrifugation. The cell suspension was counted on the counting plate, and the cells were inoculated into another 12-well culture plate coated with extracellular matrigel
  • the cells cultured under the other two culture conditions were digested, passaged and counted in the same manner as above, and the cells were inoculated according to step B or C described in step (2) of Example 4.
  • N is the number of cells at passage
  • X 0 is the number of cells at the initial seeding
  • FIG. 4 (A) and (B) of Figure 4 are the number of days of culture as the abscissa and the cell population doubling number as the ordinate.
  • the growth curves of two cases of cells under three different culture conditions are drawn using Graphpad Prism7.0 software. From Fig. 4 (A) and (B), it can be confirmed that the proliferation rate of breast epithelial cells cultured by the technique of the present invention is superior to that of the cell conditional reprogramming technique, which is similar to the organoid culture technique.
  • the cell photo in (C) of Figure 4 is a microscopic photo (under a 50-fold inverted phase contrast microscope) when HMFL-XN21 was cultured to the third generation (day 27) under three different culture conditions.
  • Fig. 5 is a comparison of the microscopic photograph when HMFL-XN12 is cultured on the 9th day with the culture medium of the present invention and the microscopic photograph when the HMFL-XN12 is cultured on the 22nd day (under a 100-fold inverted phase contrast microscope). It can be confirmed from FIG. 5 that the technology of the present invention can continuously culture primary breast epithelial cells, and the morphology of the cells after multiple passages and continuous culture does not change significantly compared with the cell morphology before passage.
  • step (1) of Example 2 was used to prepare the primary breast cancer tumor cell culture medium.
  • HMFL-XN7 Breast epithelial cells
  • the immunofluorescence method is used to detect the expression of important cancer-related biomarkers on human breast cancer tumor cells.
  • the primary antibodies used were CK8 (manufactured by Abcam), CK14 (manufactured by Abcam), ER (manufactured by Cell Signaling Technology), PR (manufactured by Cell Signaling Technology), HER2 (manufactured by Cell Signaling Technology), and Ki67 (manufactured by Cell Signaling Technology). Technology Corporation).
  • the secondary antibody used is Anti-Rabbit IgG(H+L), F(ab')2 Fragment(Alexa 488 Conjugate) (manufactured by Cell Signaling Technology), Anti-Mouse IgG(H+L), F(ab')2 Fragment(Alexa 594 Conjugate) (manufactured by Cell Signaling Technology).
  • ER and PR are important indicators for predicting whether a patient can accept endocrine therapy
  • HER2 is an important indicator for predicting whether a patient can accept anti-HER2 targeted therapy
  • Ki67 is an indicator for judging the malignancy and prognosis of breast cancer
  • CK8 and CK14 are epithelial Cell biomarkers are also used in the differential diagnosis of breast cancer.
  • the breast cancer tumor cells (HMFL-XN7) cultured to the third generation according to the technology of the present invention were inoculated on a glass slide coated with Matrigel, and the coating method was the same as that of step (2) of Example 1.
  • the primary breast epithelial cell culture medium of the present invention is used for culturing, so that the cells grow on the glass slide. After rinsing the cells twice with PBS buffer, fix the cells with 4% paraformaldehyde for 15 minutes, and then incubate them with TBST (TBS+0.1% Tween 20) containing 1% BSA and 1% Triton X-100 at room temperature The cells were washed three times with PBS buffer for one hour, each for three minutes.
  • the immunohistochemical method was used to detect the expression of important biomarkers related to breast cancer in the original tissues derived from HMFL-XN7 cells.
  • the tissues fixed with 4% paraformaldehyde were embedded in paraffin and cut into 4 ⁇ m thick tissue sections with a microtome. Subsequently, routine immunohistochemical detection was performed (see Yu et al., Science, 345(6193):216-220, 2014 for specific steps).
  • the primary antibody used is the same as that used in immunofluorescence.
  • the results of photographing the expression of ER, PR, HER2, CK8, CK14 and Ki67 and cancer-related biomarkers on the cells are shown in Figure 6(B). Among them, the results of immunohistochemistry showed that the patients had about 20% of ER(+), PR(+), HER2(-), Ki67 positive cells.
  • HMFL-XN10, HMFL-XN12 The breast cancer tumor cells (HMFL-XN10, HMFL-XN12) were continuously cultured using the same method as the technology of the present invention in step (3) of Example 4, and the mammary glands cultured in vitro and passaged 3 times Cancer tumor cells (P3) and non-passaged cancer tumor cells (P0) directly derived from breast cancer patients were collected by centrifugation, and DNeasy blood & tissue kit (manufactured by QIAGEN) was used to extract the genomic DNA of the cells. Collect 2 mL of peripheral blood from patients with cell sources, and use the same method to extract genomic DNA as a background control.
  • HMFL-XN10 is a high-frequency mutation analysis of breast cancer tumor cells from breast cancer patients who have been cultured for 3 generations using the culture method of the present invention.
  • HMFL-XN10 (P0) is the initial source of the same breast cancer patient. Analysis of isolated breast cancer tumor cells that have not been passaged. The above analysis results are produced using https://bioinfogp.cnb.csic.es/tools/venny/index.html software. The results of tumor high-frequency mutation analysis showed that the two high-frequency mutation genes were basically the same.
  • HMFL-XN12 and HMFL-XN10 cells were operated in parallel using the same method. 7(A) confirms that the breast cancer tumor cells derived from cancer tissue cultured by the technique of the present invention can maintain the original gene mutation characteristics in the patient's cancer tissue.
  • the fixed cells were dropped evenly on a glass slide and allowed to dry, and then 5 mg/ml DAPI (manufactured by Sigma) was used to stain the nuclei for 15 minutes, and the cells were photographed using a confocal laser microscope (purchased from Leica) Metaphase cleavage and paired arrangement of chromosomes.
  • Figure 7(B) shows that the chromosome morphology and number of breast cancer tumor cells cultured to the third generation of HMFL-XN10 using the technology of the present invention are normal, and there is no polyploid or hypoploid, but the chromosomes in the nucleus have duplicated two Second, and multiple chromosomes are close to each other, consistent with the karyotype characteristics of tumor cells, suggesting that the chromosomes of the cells are abnormal and have tumor cell characteristics. From Figure 7(B), it can be confirmed that the breast cancer tumor cells derived from cancer tissue cultured by the culture method of the present invention can maintain the chromosomal karyotype characteristics of breast cancer patients.
  • Example 2 Using the same method as the step (1) of Example 1 to isolate breast cancer tumor cells (HMFL-XN5, HMFL-XN29) from the cancer tissues of two patients with triple-negative breast cancer diagnosed pathologically, follow the steps of Example 2
  • the method (2) was used to culture HMFL-XN5 and HMFL-XN29 respectively.
  • the same method as the technique of the present invention in step (3) of Example 4 was used to treat breast cancer.
  • the tumor cells were digested and collected.
  • the breast cancer tumor cell culture medium of the present invention and Matrigel (registered trademark) (manufactured by BD Biotech) were mixed in a ratio of 1:1, and 100 ⁇ L of the culture medium mixed with Matrigel was absorbed.
  • NCG 6-week-old female high immunodeficiency mouse
  • the following takes a sample from a breast cancer patient surgically removed as an example to illustrate that breast cancer tumor cells cultured from a patient-derived breast cancer tumor sample can be used to detect the sensitivity of the patient's tumor cells to different drugs.
  • the plating of primary breast cancer tumor cells the breast cancer tumor cells (HMFL-XN5, HMFL-XN7, HMFL-XN10, HMFL-XN12) obtained by the same method as the technique of the present invention in step (3) of Example 4 And HMFL-XN29) single cell suspension, seeded in a 384-well plate at a density of 3000-5000 cells/well to allow the cells to adhere overnight.
  • (1) Prepare the drug storage plate by the method of concentration gradient dilution: draw 10 ⁇ L of the drug mother solution to be tested (the concentration of the drug mother solution is prepared by 2 times the maximum blood drug concentration C max of the drug in the human body), and add it to the 20 ⁇ L In the 0.5 mL EP tube of DMSO, draw 10 ⁇ L from the above EP tube to the second 0.5 mL EP tube that already contains 20 ⁇ L of DMSO, that is, dilute the medicine according to 1:3. Repeat the above method, dilute in sequence, and finally obtain the 7 concentrations required for dosing. Add different concentrations of drugs to the 384-well drug storage plate. In the solvent control group, an equal volume of DMSO was added to each well as a control.
  • the drugs to be tested are epirubicin (manufactured by MCE), lapatinib (manufactured by MCE), docetaxel (manufactured by MCE), and tamoxifen (manufactured by MCE).
  • Cell viability detection 72 hours after administration, use Cell Titer-Glo detection reagent (manufactured by Promega) to detect the chemiluminescence value of the cells after the drug is added.
  • the value of the chemiluminescence value reflects the cell viability and the effect of the drug on the cell viability Influence, add the prepared Cell Titer-Glo detection solution to each well, and use the microplate reader to detect the chemiluminescence value after mixing.
  • Figure 9 (A) ⁇ (D) respectively show the effect of two chemotherapeutic drugs epirubicin and docetaxel on breast cancer tumor cells cultured from surgically removed cancer tissue samples from five different breast cancer patients Sensitivity, sensitivity to endocrine therapy drug tamoxifen and sensitivity to targeted drug lapatinib. The results show that the cells of the same patient have different sensitivities to different drugs, and the cells of different patients have different sensitivities to the same drug.
  • breast cancer tumor cells HMFL-XN7
  • HMFL-XN12 breast cancer tumor cells derived from HER2-positive breast cancer patients
  • the test results of breast cancer tumor cells (HMFL-XN12) derived from HER2-positive breast cancer patients showed that the anti-HER2-targeted drug lapatinib was more sensitive than other patients, and half of the inhibitor was 0.92 ⁇ M.
  • a breast cancer tumor cell (HMFL-XN29) derived from a triple-negative breast cancer patient is more sensitive to the endocrine therapy drug tamoxifen and the anti-HER2 targeted drug lapatinib than other patients’ breast cancer tumor cells. Sensitivity is low.
  • the breast cancer cells (HMFL-XN5) from another triple-negative breast cancer patient were not sensitive to the four drugs tested.
  • the invention provides a culture medium and a culture method for culturing or amplifying primary breast epithelial cells in vitro.
  • the cell model obtained from the culture medium and culture method of primary mammary epithelial cells of the present invention can be used to evaluate or screen the curative effect of drugs for the treatment of breast diseases.

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Abstract

一种含有双向调节素的用于培养原代乳腺上皮细胞的培养基和使用了该培养基的培养方法。所述培养方法中，使用培养基在包被有细胞外基质胶的培养器皿上培养原代细胞，原代细胞生长在细胞外基质胶包被的培养器皿上，在原代细胞培养基所含营养因子和细胞外基质的共同作用下快速增殖。由该原代细胞培养基和原代细胞培养方法得到的细胞模型，能够用于药物的疗效评估和筛选。

Description

原代乳腺上皮细胞培养基、培养方法及其应用 技术领域
本发明属于医药技术领域，具体而言，涉及用于在体外培养或扩增原代乳腺上皮细胞的培养基及培养方法，还涉及培养得到的细胞在药物的疗效评估和筛选中的应用。
背景技术
乳腺癌是影响女性健康的最主要的恶性肿瘤之一。最新的统计结果显示，在全球范围内，乳腺癌的发病率和死亡率分别位列女性恶性肿瘤发病率和死亡率的第一位和第二位。近年来，尽管人们对乳腺癌的分子分型和发病机制的研究取得了很多进展，目前针对乳腺癌的标准药物治疗方法仍以激素类和细胞毒性类药物为主，缺乏个性化的精准用药指导。因为乳腺癌分型的多样性和复杂性以及其高度的异质性，仅凭分子或基因诊断而不辅助于功能性测试，很难真正预测临床用药的疗效(Adam A.Friedman等，Nat Rev Cancer，15(12):747-56，2015)。
功能性测试是指在体外对抗肿瘤药物在癌症患者细胞上的敏感性进行检测的方法。应用这一方法的关键在于开发生长周期短且能够代表乳腺癌患者自身生物学特性的肿瘤细胞模型。另外，所述细胞模型应操作便捷以快速高效地预测临床用药的疗效，从而及时给予癌症患者精准用药指导。然而，取自癌症患者的原代肿瘤细胞在体外建立细胞模型成功率往往很低，生长周期长，并存在成纤维细胞等间质细胞过度增殖等问题，制约着这一领域的发展。目前有两种培养原代上皮细胞/干细胞的技术在肿瘤细胞功能性测试应用领域发展得相对成熟，一种是使用经辐射的饲养细胞和ROCK激酶抑制剂Y27632来促进原代上皮细胞的生长以考察个体患者的药物敏感性的技术，即细胞条件重编程技术(Liu等，Am J Pathol，180：599-607，2012)。另一种技术是体外3D培养成体干细胞从而获得类似于组织器官的类器官技术(Hans Clevers等，Cell，11；172(1-2):373-386，2018)。
然而，这两种技术都存在一定的局限性。细胞重编程技术是一种将患者自体原代上皮细胞与鼠源性饲养细胞共培养的技术，然而，在 对患者原代细胞进行药物敏感性测试时，这些鼠源性细胞的存在会干扰患者自体原代细胞的药物敏感性检测结果；但如果撤除鼠源性饲养细胞，病人自体原代细胞就脱离了重编程环境，细胞的增殖速率和细胞内信号通路会发生明显的改变(Liu等，Am J Pathol，183(6):1862-1870，2013；Liu等，Cell Death Dis.，9(7):750，2018)，从而使患者自体原代细胞对药物的响应结果因此受到较大影响。类器官技术是将患者自体原代上皮细胞包埋在细胞外基质内进行体外三维立体培养的技术，该技术无需饲养细胞，因此不存在鼠源性饲养细胞的干扰问题，但是类器官技术的培养基内需添加多种特定的生长因子，成本昂贵，不适于普及到临床进行大规模应用。另外，类器官在整个培养过程中均需将细胞包埋在细胞外基质胶中，其细胞接种、传代和药物敏感性测试的铺板步骤相较于2D培养操作繁琐费时，且该技术所形成的类器官大小尺寸不好控制，易出现部分类器官生长过大而导致内部发生坏死的情况，因此，类器官技术相较于2D培养技术可操作性和适用性不强，需要专业技术人员操作，不适合大规模广泛应用于临床体外药物敏感性检测(Nick Barker，Nat Cell Biol，18(3):246-54，2016)。
鉴于以上技术的局限性，临床上需要开发一种原代乳腺上皮细胞培养技术，其培养周期短，成本可控，操作便捷，不受外源性细胞干扰。在将该技术应用于构建原代乳腺癌肿瘤细胞模型时，所培养的乳腺癌肿瘤细胞能代表乳腺癌患者自身的生物学特性。通过体外评估抗肿瘤药物在不同癌症患者个体所衍生的细胞模型上的敏感性，来提高临床上抗肿瘤药物的响应率，减少不合适的药物给患者造成的痛苦及医疗资源的浪费。
发明内容
本发明旨在针对现有技术的不足，提供一种用于培养原代乳腺上皮细胞的原代乳腺上皮细胞培养基以及使用该培养基的原代乳腺上皮细胞的培养方法，采用本发明的原代乳腺上皮细胞培养基和培养方法，能够达到体外培养周期短、成本可控、操作便捷并不受外源性细胞干扰的目的。在该技术应用于构建原代乳腺癌肿瘤细胞模型时，能够获 得具有乳腺癌患者自身生物学特性的原代乳腺癌肿瘤细胞，并能够应用于新药筛选和体外药物敏感性检测。
本发明的一个方面在于提供一种用于培养原代乳腺上皮细胞的原代细胞培养基，其含有双向调节素(Amphiregulin)。其中，优选双向调节素的含量为10ng/ml以上，从成本上考虑，优选为10ng/ml～100ng/ml。
本发明的原代细胞培养基优选还含有：表皮生长因子(EGF)、胰岛素(Insulin)、B27、ROCK激酶抑制剂Y27632、神经调节素1(Neuregulin1)、成纤维细胞生长因子7(FGF7)、TGFβI型受体抑制剂A8301和P38/MAPK抑制剂SB202190中的一种或多种或全部。其中，优选：所述EGF的含量为2.5ng/ml～20ng/ml；胰岛素的含量为1μg/ml～10μg/ml；所述B27以1:25～1:100的终浓度稀释；所述Y27632含量为5μM～15μM；所述神经调节素1的含量为5nM～20nM；所述FGF7的含量为2.5ng/ml～20ng/ml；所述A8301的含量为100nM～500nM；所述SB202190的含量为100nM～500nM。
该培养基配方成分与细胞条件重编程培养基和乳腺上皮细胞类器官培养基成分相比，添加了双向调节素，不包含血清、牛垂体提取物等不确定成分，也不包含Wnt激动剂、R-spondin家族蛋白、BMP抑制剂等类器官培养所必须的龛因子，并且不包含成纤维细胞生长因子10(FGF10)、烟酰胺(Nicotinamide)和N-乙酰半胱氨酸(N-Acetylcysteine)，从而大大降低了培养基的成本，简化了配制培养基的操作流程，实现了成本可控和操作便捷的原代乳腺上皮细胞的体外培养。
本发明中，原代乳腺上皮细胞可以为乳腺癌肿瘤细胞、正常乳腺上皮细胞、乳腺上皮干细胞。
本发明的一个方面在于提供一种原代乳腺上皮细胞的培养方法，其包括以下步骤：
(1)按上述配方配制本发明的原代细胞培养基。
(2)用细胞外基质胶稀释液包被培养器皿。
其中，该细胞外基质胶使用低生长因子型细胞外基质胶，例如，可采用市售的Matrigel(Corning：354230)或BME(Trevigen： 3533-010-02)。更具体而言，用无血清的培养基稀释细胞外基质胶，培养基可以是本发明的原代细胞培养基，也可以是DMEM/F12(Corning：R10-092-CV)。细胞外基质胶的稀释比例为1:50-400，优选为1:100-200。包被方法为将稀释后的细胞外基质胶加入培养器皿内，使其完全覆盖培养器皿底部，静置包被30分钟以上，优选在37℃条件下静置包被，优选包被30～60分钟。包被结束后吸弃多余的细胞外基质胶稀释液，培养器皿备用。
(3)从乳腺组织分离得到原代乳腺上皮细胞。
原代乳腺上皮细胞例如可以来源于乳腺癌组织样本和癌旁组织样本。乳腺癌组织样本例如来源于进行过说明并获得同意的乳腺肿瘤患者手术切除癌组织样本，癌旁组织样本采集自离乳腺癌组织距离至少5cm以上的乳腺组织。在患者手术切除或活检后的半小时内进行上述组织样本的收集。更具体而言，在无菌环境下，切取非坏死部位的组织样本，其体积在0.5cm 3以上，将其置于预冷的10-50mL DMEM/F12培养基中，培养基盛在塑料无菌带盖离心管内，冰上运输至实验室；其中，DMEM/F12培养基中含有50-200U/mL(例如100U/mL)青霉素和50-200U/mL(例如100U/mL)链霉素(以下简称运输液)。
在生物安全柜内，将组织样本转移至细胞培养皿内，用运输液润洗组织样本，将组织样本表面的血细胞清洗掉，并剔除组织样本表面的皮肤、筋膜等不需要的组织。
将润洗后的组织样本转移至另一个新的培养皿内，加入5-25mL运输液，用无菌手术刀片和手术镊将组织样本分割为直径小于1mm 3的组织碎块。
将组织样本碎块转移至离心管内，用台式离心机以至少1000转/分钟离心3-10分钟；然后用移液器小心移除离心管内上清，再用5-25mL含胶原酶II(0.5-5mg/mL，例如1mg/mL)和胶原酶IV(0.5-5mg/mL，例如1mg/mL)的无血清DMEM/F12培养基重悬，置37℃恒温摇床上进行振荡消化，时间为至少1小时(消化时间取决于样本大小；如果样本大于1g，则消化时间增至1.5-2小时)；之后用台式离心机以至少300g/分钟离心3-10分钟，弃去上清液，消化后的组织细胞用5-25mL含例如10％小牛血清的DMEM/F12培养基重悬，研磨过筛，细胞筛孔 径为例如100μm，将过筛的细胞悬液收集于离心管中；用血细胞计数板计数。
然后将细胞悬液在离心机中以至少300g/分钟离心3-10分钟，弃去上清，再用本发明的原代细胞培养基重悬。
(4)在包被好的培养器皿内接种步骤(3)中分离得到的原代乳腺上皮细胞。
更具体而言，在T12.5培养瓶中按1×10 3～1×10 5细胞/mL(例如1×10 4细胞/mL)的密度接种原代乳腺癌肿瘤细胞，加入1-10mL原代上皮细胞培养基，在例如37℃、5％CO 2的条件下于细胞培养箱中培养8-10天，氧气浓度可为常氧(15-20％氧气浓度)，也可为低氧(0.5～4％氧气浓度)，期间每4天换成新鲜的原代细胞培养基，在原代乳腺上皮细胞长至占培养瓶底面积80％～90％左右的细胞密度时进行消化传代。
该接种步骤无需使用饲养细胞，相比细胞条件重编程技术，免去了培养和辐照饲养细胞的操作步骤。该步骤相比类器官技术，也无需在冰上将原代细胞和基质胶混匀后形成胶滴，并等待胶滴凝固后加入培养基，预先包被好的培养器皿可直接用于原代细胞接种，此外，包被培养器皿仅需少量稀释后的细胞外基质胶，相比类器官技术，节约了价格昂贵的细胞外基质胶的使用量，也简化了操作步骤。
(5)任选地，接种后的原代乳腺上皮细胞在培养4～10天后，当培养瓶内形成的细胞克隆最大直径达500μm时，弃去上清，加入1-2mL 0.05％胰酶(Thermo Fisher：25300062)进行细胞消化，室温下孵育5-20分钟；然后用含有例如10％(v/v)小牛血清、100U/mL青霉素和100U/mL链霉素的培养液1-10mL重悬消化处理后的细胞，以至少300g/分钟离心3-10分钟，使用本发明的原代细胞培养基将消化后的单细胞重悬，将所得到的细胞悬液置入包被有细胞外基质胶的T25细胞培养瓶中继续扩大培养。T25细胞培养瓶的包被操作同步骤(2)。
扩增的乳腺上皮细胞呈2D生长，避免了类器官技术扩增出现的类器官大小不均一和生长过大的类器官出现内部坏死等情况。
另外，由本发明的原代乳腺上皮细胞的培养方法培养得到的乳腺上皮细胞、特别是乳腺癌肿瘤细胞能够用于药物的疗效评估和筛选， 包括以下步骤：
(1)获取原代乳腺上皮细胞，特别优选获取源自乳腺癌患者的癌组织样本或活检癌组织样本，分离得到原代乳腺上皮细胞，根据如上所述的方法培养并扩增原代乳腺上皮细胞(特别是原代乳腺癌肿瘤细胞)达至少10 5数量级、优选至少10 6数量级的细胞数目。
(2)选定需要检测的药物。
(3)药物以其最大血浆浓度C max为参考，以2-5倍C max为起始浓度，稀释多个不同的药物浓度梯度，例如5-10个、优选6-8个药物浓度梯度。
(4)将步骤(1)中培养得到的乳腺上皮消化成单细胞悬液，用血细胞计数板进行计数，用含有细胞外基质胶的本发明的原代细胞培养基将单细胞悬液稀释，按每孔1000-10000个的密度将稀释后的细胞悬液均匀地加入到多孔板内，例如每孔50μL细胞稀释液，并进行过夜贴壁。
该步骤避免了细胞重编程技术出现的由于饲养细胞的存在而干扰原代细胞计数和后续的原代细胞活力检测的问题，也无需像类器官技术一样，在冰上将细胞悬液与基质胶混合包埋再铺板的繁琐步骤，从而大大简化了操作流程，增强了技术的可操作性和实用性。由于接种的细胞为单细胞悬液而不是像类器官一样的3D结构，所以该技术与类器官技术相比，铺板的细胞数更加均一，铺板产生的孔间细胞数差异小，也更适合进行后续的高通量药物筛选操作。
(5)采用高通量自动化工作站，对步骤(4)中得到的贴壁细胞添加梯度稀释后的所选定的传统化疗药物、靶向药物、抗体药物或几种药物组合等候选药物，进行处理。
(6)加药处理数小时后，例如72小时后，采用Cell-Titer Glo发光法细胞活力检测试剂盒(Promega：G7573)检测乳腺上皮细胞的存活率，进行药物活性筛选。
具体而言，向每孔加入例如50μL Cell Titer-Glo试剂(Promega：G7573)，均匀震荡后，用荧光酶标仪测量各孔的化学发光强度，根据测得的数值，以药物浓度为横坐标，荧光强度为纵坐标，应用GraphPad Prism 7.0软件绘制药物量-效曲线，计算各个药物对所测试细胞的增殖 的抑制强度。
在本发明的原代乳腺癌肿瘤细胞在药物筛选和体外药物敏感性检测的应用中，由于不是细胞共培养体系，所以不会出现细胞重编程技术中的饲养细胞干扰检测结果的现象。由于细胞呈2D生长，与药物的作用时间也比类器官技术的药物检测时间短(类器官技术的平均给药时间为6天)。
本发明的有益效果还包括：
(1)提高原代乳腺上皮细胞培养的成功率，成功率达到90％以上；
(2)保证体外原代培养的乳腺上皮细胞能够再现原代细胞来源病人的病理表型和异质性；
(3)所培养的原代乳腺上皮细胞不受成纤维细胞、脂肪细胞等间质细胞的干扰，能得到纯化的乳腺上皮细胞；
(4)培养基成分不含血清，所以不受不同批次血清质量和数量的影响；
(5)扩增乳腺上皮细胞效率高，只要有10 4级别的细胞数量就可在两周左右时间内成功扩增出10 6数量级的乳腺上皮细胞，扩增出的乳腺上皮细胞还可以连续传代；
(6)传代步骤无需冰上操作和解离基质胶，10-15分钟内即可完成细胞的消化传代；
(7)培养成本可控：原代乳腺癌培养基无需加入价格昂贵的Wnt激动剂、R-spondin家族蛋白、BMP抑制剂、FGF10等因子，是对已有原代乳腺上皮细胞类器官培养基的简化和改进，细胞接种也无需使用浓度较高的细胞外基质与原代细胞混合形成胶滴，而只需使用少量细胞外基质胶制备的稀释液，节约了成本较高的细胞外基质的用量；
(8)操作便捷，该技术相比条件重编程技术，无需培养饲养细胞并对饲养细胞进行辐射，避免了不同批次饲养细胞的质量和数量影响原代细胞培养效率的问题，药物筛选的铺板和检测的对象只有原代乳腺上皮细胞，而不受细胞条件重编程技术所述的共培养体系中饲养细胞的干扰；相比类器官技术，本发明采用的细胞外基质胶的包被方法，培养器皿可预先准备，无需像类器官技术一样将细胞包埋于基质胶内，所述技术操作步骤简便易行；
(9)所述技术培养获得的乳腺上皮细胞数量大，均一化程度高，适合高通量筛选新候选化合物和为病人提供高通量药物体外敏感性功能测试。
采用本实施方式的细胞培养基，可培养来源于包括人的或其他哺乳动物的乳腺上皮细胞，包括乳腺癌肿瘤细胞、正常乳腺上皮细胞、乳腺上皮干细胞、或者包含这些细胞中的至少任一种的组织。
此外，还可从所述细胞和所述组织中的至少一方形成类器官。
另外，通过本实施方式的培养方法获得的细胞可应用于再生医疗、乳腺上皮细胞的基础医学研究、药物应答的筛选、以及来源于乳腺疾病的新药研发等。
附图说明
图1为用本发明的原代细胞培养方法对临床乳腺组织样本分离得到的细胞进行培养所获得的原代乳腺上皮细胞的倒置相差显微镜下照片。
图2是将从两例不同的乳腺癌临床组织样本(HMFL-XN30，HMFL-XN22)分离得到的原代乳腺癌肿瘤细胞分别接种在经过细胞外基质胶包被和不经过任何包被的培养板内培养得到的细胞镜下照片。
图3是用于说明双向调节素对原代乳腺癌肿瘤细胞的促增殖作用的效果。
图4是将从两例乳腺癌临床组织样本(HMFL-XN12，HMFL-XN21)分离得到的细胞分别采用细胞条件重编程技术、本发明技术和类器官技术培养所获得的细胞生长曲线对比图和培养至第27天时HMFL-XN21的镜下照片。
图5是将从一例乳腺癌临床组织样本(HMFL-XN12)分离得到的细胞采用本发明技术培养至第9天和培养至第22天的乳腺癌肿瘤细胞在倒置显微镜下的照片。
图6是从一例乳腺癌手术切除样本分离得到的细胞(HMFL-XN7)采用本发明技术培养获得的乳腺癌肿瘤细胞的免疫荧光染色结果与该组织样本自身原始的组织切片免疫组化结果对比。
图7是从两例乳腺癌手术切除样本分离得到的细胞采用本发明技 术培养获得的不同代数的乳腺癌肿瘤细胞的基因突变一致性分析和其中一例的染色体核型分析结果。
图8是对来源于两例病理诊断为三阴性乳腺癌患者的癌组织的原代乳腺癌肿瘤细胞采用本发明技术进行培养所获得的乳腺癌肿瘤细胞在小鼠体内的成瘤情况。
图9是表示采用本发明技术培养的原代乳腺癌肿瘤细胞对不同化疗药物和靶向药物的剂量-效应曲线及经计算得到的半数抑制率。
具体实施方式
[实施例1]
人原代乳腺上皮细胞的分离和原代乳腺上皮细胞培养基的优化
(1)人原代乳腺上皮细胞的分离
乳腺癌组织样本来源于五例进行过说明并获得同意的乳腺肿瘤患者手术切除癌组织样本，它们分别是HMFL-XN1，HMFL-XN3，HMFL-XN4，HMFL-XN6，HMFL-XN8。下面以其中一例样本(HMFL-XN1)进行说明。在患者手术切除或活检后的半小时内进行上述组织样本的收集。更具体而言，在无菌环境下，切取非坏死部位的组织样本，其体积在0.5cm 3以上，将其置于预冷的20mL DMEM/F12培养基(Corning公司制)中，培养基盛在50mL塑料无菌带盖离心管内，冰上运输至实验室；其中，DMEM/F12培养基中含有100U/mL青霉素和100U/mL链霉素(以下简称运输液)。
在生物安全柜内，将组织样本(HMFL-XN1)转移至100mm细胞培养皿内，用运输液润洗组织样本，将组织样本表面的血细胞清洗掉，并剔除组织样本表面的皮肤、筋膜等不需要的组织。
将润洗后的组织样本转移至另一个新的100mm培养皿内，加入10mL运输液，用无菌手术刀片和手术镊将组织样本分割为直径小于1mm 3的组织碎块。
将组织样本碎块转移至50mL离心管内，用台式离心机以1200转/分钟离心5分钟；然后用移液器小心移除离心管内上清，再用10mL含胶原酶II(1mg/mL)和胶原酶IV(1mg/mL)的无血清DMEM/F12培养基重悬，置37℃恒温摇床上进行振荡消化，时间为1小时；之后 用台式离心机以350g/分钟离心5分钟，弃去上清液，消化后的组织细胞用10mL含10％小牛血清的DMEM/F12培养基重悬，研磨过筛，细胞筛孔径为100μm，将过筛的细胞悬液收集于50mL离心管中；用血细胞计数板计数。
然后将细胞悬液在离心机中以350g/分钟离心5分钟，弃去上清，再用本发明的原代细胞培养基重悬。
另外四例乳腺肿瘤组织样本按照以上同样的方法进行分离。
(2)原代乳腺上皮细胞培养基的优化
将Matrigel(注册商标)(BD生物科技公司制)使用无血清DMEM/F12培养基按1:100稀释比例，配制成细胞外基质稀释液，在48孔培养板内加入200μl/孔的细胞外基质稀释液使其完全覆盖培养板孔的底部。在37℃培养箱内静置1小时。1小时后，移除细胞外基质稀释液，得到包被有Matrigel的培养板。
首先配置基础培养基。向市售的DMEM/F-12培养基中以说明书浓度(1:100倍稀释)加入GlutaMAX-I(Thermo Fisher SCIENTIFIC公司制)，以最终浓度10μg/ml的条件添加人胰岛素(Sigma公司制)，以最终浓度10μM的条件添加ROCK激酶抑制剂Y27632(Sigma公司制)，以1:100倍稀释比例加入青霉素-链霉素(Thermo Fisher SCIENTIFIC公司制)配置得到基础培养基。
接着，在基础培养基内分别加入不同种类的添加剂(表1)配置成含有不同添加成分的乳腺上皮细胞培养基，将不同成分的培养基按500μl/孔体积加入至包被有细胞外基质胶(Matrigel)的48孔板内。将本实施例步骤(1)从乳腺癌组织分离得到的乳腺癌肿瘤细胞(HMFL-XN1)以1×10 4个/孔的细胞密度接种在Matrigel包被过的48孔培养板内，以37℃、5％CO 2浓度、20％氧浓度的条件，使等数目的新鲜分离的乳腺癌肿瘤细胞(HMFL-XN1)在不同的培养基配方条件下进行培养。培养开始后每4天进行一次培养基的更换。培养10天后，进行细胞计数。其中，作为实验对照，使用未添加任何添加剂的基础培养基。其余四例乳腺癌组织样本分离得到的乳腺癌肿瘤细胞按照以上同样的方法进行培养和计数。将实验结果示于表1。
[表1]
Figure PCTCN2019119116-appb-000001
其中，“+”表示相比基础培养基，加入该添加剂的培养基对从乳腺癌组织分离出的原代乳腺癌肿瘤上皮细胞中的至少三例有促进增殖的作用；“－”表示添加该添加剂的培养基对从乳腺癌组织分离出的原代乳腺癌肿瘤上皮细胞中的至少两例显示有抑制增殖的作用；“○”表示添加该添加剂的培养基对从乳腺癌组织分离出的原代乳腺癌肿瘤上皮细胞中的至少三例的增殖没有明显的影响。
[实施例2]
人原代乳腺上皮细胞的培养
(1)原代乳腺上皮细胞培养基的制备
首先，按照实施例1的步骤(2)同样的方法制备基础培养基。向基础培养基中以最终浓度20ng/ml的条件添加人双向调节素(R&D systems公司制)，以最终浓度10ng/ml的条件添加EGF(Peprotech公司制)，以1:50比例稀释条件添加B27(Thermo Fisher SCIENTIFIC公司制)，以最终浓度10nM的条件添加人神经调节素1(Peprotech公司制)，以最终浓度10ng/ml添加FGF7(R&D systems公司制)，以最终浓度500nM条件添加TGFβ1抑制剂A8301(MCE公司制)，以最终浓度500nM条件添加P38/MAPK抑制剂SB202190(MCE公司制)，制备原代乳腺上皮细胞培养基。
(2)乳腺癌组织来源的原代乳腺癌肿瘤细胞和癌旁组织来源的乳腺正常上皮细胞的培养
使用与实施例1的步骤(1)同样的方法分别从同一乳腺癌患者的癌组织和癌旁乳腺组织分离获得癌组织来源的乳腺上皮细胞及癌旁组织来源的乳腺上皮细胞。其中，癌旁乳腺组织样本采集自离乳腺癌组织距离至少5cm以上的乳腺组织。接着，将癌组织来源的乳腺癌肿瘤细胞用血细胞计数板进行计数，然后按4×10 4个/孔密度接种至Matrigel(注册商标)(BD生物科技公司制)包被处理过的12孔板内。向12孔板中添加2mL制备好的原代乳腺上皮细胞培养基，在37℃以5％CO 2浓度、20％氧浓度的条件进行培养。
图1的(A)是从一例临床手术切除的乳腺肿瘤样本分离的乳腺癌肿瘤细胞(HMFL-XN11)自接种开始后培养至第7天的镜下照片(50倍倒置相差显微镜下)。镜下观察可见，所培养的癌组织来源的原代乳腺癌肿瘤细胞纯度较高，不含有成纤维细胞等间质细胞。图1的(B)是从与HMFL-XN11来源相同的病人的癌旁组织样本分离的原代乳腺正常上皮细胞(HMFL-XN11-N)按4×10 4个/孔密度接种至Matrigel包被处理过的12孔板，自接种后培养至第7天的镜下照片(50倍倒置相差显微镜下)。图1的(A)和(B)说明采用本发明的原代乳腺上皮细胞培养基和培养方法对乳腺癌组织来源的乳腺癌肿瘤细胞和癌旁组织来源的乳腺正常上皮细胞均可以实现高效的体外培养。培养出的原代细胞均不含成纤维细胞等间质细胞。
图1的(C)和(D)是另一例从临床手术切除的乳腺癌组织样本 分离的原代乳腺癌肿瘤细胞(HMFL-XN15)按4×10 4个/孔密度接种至Matrigel包被处理过的12孔板，自接种后培养第4天的镜下照片和自接种后培养第10天的镜下照片对比(100倍倒置相差显微镜下)。其中，图1的(D)表示采用本发明的原代细胞培养基和培养方法可培养出构成乳腺的两种不同的细胞亚群：管腔细胞和肌上皮细胞，实现了保持肿瘤样本异质性的体外培养。对比图1的(C)和(D)可确认采用本发明的培养基和培养方法对乳腺癌组织样本分离、接种和培养，在接种并培养的第4天即可有明显的乳腺上皮细胞克隆形成，培养至第10天时，细胞数目实现了指数级的扩增，提示本发明技术是一种高效的体外扩增乳腺上皮细胞的技术。
(3)原代乳腺上皮细胞在正常氧气浓度条件和低氧条件下的培养
使用与实施例1的步骤(1)同样的方法从一例乳腺癌患者的癌组织中分离获得乳腺癌组织来源的原代乳腺癌肿瘤细胞(HMFL-XN30)。接着，将等细胞数目(4×10 4个/孔)的原代乳腺癌肿瘤细胞HMFL-XN30接种至Matrigel(注册商标)(BD生物科技公司制)包被处理过的12孔板。向12孔板中添加2mL制备好的原代乳腺上皮细胞培养基，分别以氧浓度20％的条件(正常氧气浓度条件)和氧浓度2％的条件(低氧条件)对等数目的乳腺癌肿瘤细胞进行培养。培养至第7天时的镜下照片(100倍倒置相差显微镜下)。图1的(E)和(F)是癌组织来源的乳腺上皮细胞分别在常氧和低氧条件下培养所得到的细胞镜下的对比。由图1(E)和(F)可确认，采用本发明的培养基和培养方法既可以在常氧条件下(20％氧浓度)也可以在低氧条件下(2％氧浓度)对癌组织来源的原代乳腺上皮细胞进行高效培养。
(4)乳腺癌组织来源的原代乳腺癌肿瘤细胞在包被细胞外基质胶和不包被细胞外基质胶的条件下培养效果的比较
使用与实施例1的步骤(1)同样的方法从两例乳腺癌患者的癌组织中分离获得癌组织来源的原代乳腺癌肿瘤细胞(HMFL-XN30，HMFL-XN22)。接着，将等数目(4×10 4个/孔)的原代乳腺癌肿瘤细胞HMFL-XN30分别接种至Matrigel(注册商标)(BD生物科技公司制)包被处理过的12孔板和未经过任何处理的12孔板中。向12孔板中添加2mL制备好的原代乳腺上皮细胞培养基，以氧浓度20％的条件 对等数目的原代乳腺癌肿瘤细胞进行培养。培养10天后，进行拍照。HMFL-XN22(P3)为HMFL-XN22自分离后分别在包被Matrigel和不包被Matrigel的条件下连续培养至第三代的乳腺癌肿瘤细胞，其余步骤和HMFL-XN30操作步骤相同。
图2是乳腺癌组织源性原代乳腺癌肿瘤细胞HMFL-XN30和HMFL-XN22(P3)分别在包被Matrigel和不包被Matrigel的条件下培养至第10天的细胞的镜下照片。根据图2可以确认，采用Matrigel包被处理培养板较未经过任何处理的培养板，更有利于乳腺癌肿瘤细胞的增殖。
[实施例3]
双向调节素对乳腺癌组织来源的原代乳腺癌肿瘤细胞的促增殖效果
(1)使用与实施例2的步骤(1)同样的方法制备原代乳腺上皮细胞培养基。另外，在原代乳腺上皮细胞培养基的配方中去除双向调节素，配制另一种不含双向调节素的原代培养基。
(2)使用与实施例1的步骤(1)同样的方法获得两例不同的乳腺癌患者手术切除癌组织样本来源的原代乳腺癌肿瘤细胞(HMFL-XN2，HMFL-XN12)和一例乳腺癌患者穿刺样本的原代乳腺癌肿瘤细胞(HMFL-XN13)。
将手术切除癌组织来源的原代乳腺癌肿瘤细胞(HMFL-XN2)按照同样的接种密度(5×10 4个/孔)分别接种至Matrigel(注册商标)(BD生物科技公司制)包被处理过的12孔板内。使用含双向调节素的本发明原代乳腺上皮细胞培养基和不含双向调节素的原代培养基在37℃以氧浓度20％的条件分别对细胞进行培养，每组两个复孔。图3的(A)为将源自同一例乳腺癌患者所分离的细胞(HMFL-XN2)按照等细胞数目(5×10 4个/孔)接种，在不含双向调节素和含双向调节素的原代培养基培养6天后所形成的肿瘤细胞克隆的显微镜照片(100倍倒置相差显微镜下)。
图3的(B)为三例乳腺癌组织来源的乳腺癌肿瘤细胞(HMFL-XN2，HMFL-XN-12和HMFL-XN13)分别使用不含双向调节素和含双向调节素的原代培养基培养10天后的细胞计数结果，每组 两个复孔。由图4可知，以不含双向调节素的培养基组所培养出的细胞作为对照，添加双向调节素能够促进原代乳腺上皮细胞的增殖，添加该成分可增加至少50％、高达250％的促进原代乳腺上皮细胞增殖的效果。
[实施例4]
乳腺癌组织来源的原代乳腺癌肿瘤细胞的持续培养及生长曲线绘制
(1)使用与实施例2的步骤(1)同样的方法制备原代乳腺上皮细胞培养基。作为对照，另外制备细胞条件重编程技术用培养基(以下也称“细胞条件重编程技术培养基”)，配制步骤参见(Liu等，Nat Protoc.，12(2):439-451，2017)，培养基配方见表2。此外，作为另一对照例，制备乳腺类器官的培养基(以下也称“类器官技术培养基”)，配制步骤参见(Hans Clevers等，Cell，11；172(1-2):373-386，2018)，培养基配方见表3。
[表2]
细胞条件重编程技术培养基成分
培养基成分 供应商 货号 终浓度
DMEM培养基 Corning 10-013-CVR 65％
胎牛血清 Gibico 16140-071 10％
Ham’s F12营养液 Gibico 11765-054 25％
氢化可的松 Sigma-Aldrich H-0888 25ng/ml
表皮生长因子 R&D 236-EG 0.125ng/ml
胰岛素 Sigma-Aldrich I-5500 5μg/ml
两性霉素B Sigma-Aldrich V900919 250ng/ml
庆大霉素 Gibico 15710-064 10μg/ml
霍乱毒素 Sigma-Aldrich C8052 0.1nM
Y27632 Enzo 270-333M025 10μM
[表3]
类器官技术培养基成分
培养基成分 供应商 货号 终浓度
R-Spondin 1条件培养基 自制 / 10％
或R-Spondin 3重组蛋白 R&D 3500-RS/CF 250ng/ml
神经调节素1 Peprotech 100-03 5nM
成纤维细胞生长因子7 Peprotech 100-19 5ng/ml
成纤维细胞生长因子10 Peprotech 100-26 20ng/ml -
表皮生长因子 Peprotech AF-100-15 5ng/ml
Noggin Peprotech 120-10C 100ng/ml
A83-01 Tocris 2939 500nM
Y-27632 Abmole Y-27632 5mM
SB202190 Sigma S7067 500nM
B27 Gibco 17504-44 1x
N-乙酰半胱氨酸 Sigma A9165-5g 1.25mM
烟酰胺 Sigma N0636 5mM
谷氨酰胺添加剂100x Invitrogen 12634-034 1x
Hepes缓冲液 Invitrogen 15630-056 10mM
青霉素/链霉素 Invitrogen 15140-122 100U/ml
Primocin Invivogen Ant-pm-1 50mg/ml
高级DMEM/F12培养基 Invitrogen 12634-034 1x
(2)使用与实施例1的步骤(1)同样的方法获得两例乳腺癌组织来源的原代乳腺癌肿瘤细胞(HMFL-XN12和HMFL-XN21)。接着，HMFL-XN12和HMFL-XN21按照相同的密度(4×10 4个/孔)分别在以下三种培养条件下培养：
A.本发明技术：按4×10 4个/孔接种密度将原代乳腺癌肿瘤细胞接种至Matrigel(注册商标)(BD生物科技公司制)包被处理过的12孔板内，采用2mL的本发明的原代乳腺上皮细胞培养基进行培养；
B.细胞条件重编程技术：按4×10 4个/孔接种密度将原代乳腺癌肿瘤细胞接种至铺有受γ射线辐照过后的小鼠成纤维细胞系J2细胞(购自Kerafast公司)上，采用细胞条件重编程技术培养基在12孔板中进行培养(具体步骤参见Liu等，Am J Pathol，183(6):1862-1870，2013)；
C.按4×10 4个/孔接种密度将原代乳腺癌肿瘤细胞接种至Matrigel(注册商标)(BD生物科技公司制)包被处理过的12孔板内，采用2mL的类器官技术培养基在12孔板中进行培养。
上述三种培养中，每4天对三种培养条件下培养的细胞进行换液。
(3)对于采用本发明技术培养的原代乳腺癌肿瘤细胞 (HMFL-XN12和HMFL-XN21)，分别在培养板内生长达到约80％板底面积时，弃去原12孔板内的培养基上清，加入1mL 0.05％胰酶(Thermo Fisher：25300062)对细胞进行消化，37℃下孵育15分钟，15分钟后，用含有10％(v/v)小牛血清、100U/mL青霉素和100U/mL链霉素的DMEM/F12培养液5mL重悬消化处理后的细胞并收集至离心管内，以350g/分钟转速离心5分钟，使用本发明的培养基重悬离心后的细胞沉淀，细胞计数板对细胞悬液进行计数，按4×10 4个/孔密度将细胞接种置另一包被有细胞外基质胶的12孔培养板中继续培养。
另两种培养条件下培养的细胞采用如上述同样的操作方式进行消化传代和计数，并按照实施例4的步骤(2)中所述的B或C步骤分别进行细胞接种。
当传代后的细胞在培养板内生长再次达到约80％板底面积时，再次按上述操作方法消化收集所培养获得的细胞并计数。同样按4×10 4个/孔密度接种并持续培养。
以下为原代乳腺上皮细胞在不同技术培养条件下细胞的群体倍增数的计算公式：
细胞群体倍增数＝[log(N/X 0)]/log2
其中，N为传代时的细胞数目，X 0为初始接种时的细胞数目(参见Greenwood等，Environ Mol Mutagen 2004，43(1):36-44)。
图4的(A)和(B)是以培养天数为横坐标，细胞群体倍增数为纵坐标，采用Graphpad Prism7.0软件绘制的三种不同技术培养条件下的两例细胞的生长曲线。由图4的(A)和(B)可以确认本发明技术培养的乳腺上皮细胞的增殖速度优于细胞条件重编程技术，和类器官培养技术相近。
图4的(C)的细胞照片为HMFL-XN21在三种不同的培养条件下培养至第三代(第27天)时的镜下照片(50倍倒置相差显微镜下)。
图5是对HMFL-XN12采用本发明的培养基培养培养至第9天时的镜下照片和培养至第22天时的镜下照片对比(100倍倒置相差显微镜下)。由图5可以确认本发明技术可以对原代乳腺上皮细胞进行持续培养，且多次传代并持续培养的细胞形态和未传代前的细胞形态相比，未发生明显改变。
[实施例5]
癌组织来源的原代乳腺癌肿瘤细胞免疫标记物的鉴定
(1)使用与实施例2的步骤(1)同样的方法制备原代乳腺癌肿瘤细胞培养基。
(2)从一例乳腺癌患者的临床手术切除样本取出约黄豆粒大小的癌组织，浸泡在10mL 4％多聚甲醛中固定。剩余癌组织使用与实施例1的步骤(1)同样的方法获得乳腺上皮细胞(HMFL-XN7)。使用实施例4的步骤(3)中本发明技术同样的方法将HMFL-XN7持续培养至第三代。
(3)采用免疫荧光法检测人乳腺癌肿瘤细胞上与癌症相关的重要生物标记物的表达。所用的一抗为CK8(Abcam公司制)，CK14(Abcam公司制)，ER(Cell Signaling Technology公司制)，PR(Cell Signaling Technology公司制)，HER2(Cell Signaling Technology公司制)，Ki67(Cell Signaling Technology公司制)。所用的二抗为Anti-Rabbit IgG(H+L),F(ab')2 Fragment(Alexa
Figure PCTCN2019119116-appb-000002
488 Conjugate)(Cell Signaling Technology公司制)，Anti-Mouse IgG(H+L),F(ab’)2 Fragment(Alexa
Figure PCTCN2019119116-appb-000003
594 Conjugate)(Cell Signaling Technology公司制)。其中，ER、PR是预测患者是否可接受内分泌治疗的重要指标；HER2是预测患者是否可接受抗HER2靶向治疗的重要指标；Ki67是判断乳腺癌恶性程度和预后的指标；CK8和CK14是上皮细胞的生物标志物，也用于乳腺癌的鉴别诊断。
将按本发明技术培养至第三代的乳腺癌肿瘤细胞(HMFL-XN7)接种于包被有Matrigel的载玻片上，包被方式同实施例1的步骤(2)。采用本发明的原代乳腺上皮细胞培养基进行培养，使细胞在载玻片上生长。用PBS缓冲液润洗细胞2次后，采用4％多聚甲醛将细胞固定15分钟，随后用含1％BSA、1％Triton X-100的TBST(TBS+0.1％Tween 20)在室温下孵育细胞一个小时，PBS缓冲液冲洗3次，每次3分钟。除去PBS液，在载玻片上滴加50μL一抗稀释液(按抗体说明书配置)，室温下孵育60分钟，PBS冲洗三次，每次3分钟；滴加二抗稀释液(按抗体说明书配置)，室温下孵育60分钟，PBS冲洗三次，每次3分钟。用1μg/mL的DAPI染料(Sigma公司制)孵育细胞10 分钟。用PBS漂洗细胞一次。用一滴封片剂(Thermo Fisher Scientific公司制)封闭盖玻片后，拍摄细胞上ER、PR、HER2、CK8、CK14和Ki67这些与乳腺癌相关的生物标记物的表达(400倍荧光显微镜下)。结果如图6的(A)所示。免疫荧光染色结果显示采用本发明技术培养至第三代的乳腺上皮细胞为ER(+)、PR(+)、HER2(-)、Ki67阳性细胞数约为20％。
(4)采用免疫组化法检测HMFL-XN7细胞来源的原始组织内与乳腺癌相关的重要生物标记物的表达。4％多聚甲醛固定后的组织，经石蜡包埋，用切片机切成4μm厚的组织切片。随后进行常规的免疫组织化学检测(具体步骤参见Yu等，Science，345(6193):216-220，2014)。所使用的一抗同免疫荧光法所用的一抗。拍摄细胞上ER、PR、HER2、CK8、CK14和Ki67与癌症相关生物标记物的表达(200倍生物显微镜下)结果如图6的(B)所示。其中，免疫组化结果显示患者为ER(+)、PR(+)、HER2(-)、Ki67阳性细胞数约20％。
由图6的(A)和(B)可以确认，采用本发明技术培养的乳腺癌肿瘤细胞(HMFL-XN7)培养至第三代时细胞上与乳腺癌相关的生物标记物的表达情况与细胞来源的原始组织切片的标记物表达情况一致。说明采用本发明技术所培养的细胞保持了乳腺癌病人癌组织的原始病理特性。
[实施例6]
癌组织来源的原代乳腺癌肿瘤细胞的基因突变及核型分析
(1)基因突变分析：采用实施例4的步骤(3)中本发明技术同样的方法将乳腺癌肿瘤细胞(HMFL-XN10，HMFL-XN12)进行持续培养，将体外培养并传代3次的乳腺癌肿瘤细胞(P3)和乳腺癌病人直接来源的未经过传代的癌肿瘤细胞(P0)离心收集后，使用DNeasy blood&tissue kit(QIAGEN公司制)提取细胞的基因组DNA。收集细胞来源患者的外周血2mL，采用同样的方法提取基因组DNA作为背景对照。随后，对细胞和血液样本的基因组DNA进行全外显子组测序(具体操作步骤参见Hans Clevers等，Cell，11；172(1-2):373-386，2018)，并对测序结果进行肿瘤高频突变基因分析。使用MuSiC软件进行肿瘤高频突变分析，MuSiC以该肿瘤样本来源的患者外周血基因 突变为背景，对基因上的各个突变类型进行统计检验，检测出显著高于背景突变率的基因。所得分析结果如图7的(A)所示。图7的(A)中维恩图显示采用本发明技术培养获得的不同代数的乳腺癌肿瘤细胞所携带的高频基因突变数目的比对。HMFL-XN10(P3)是对用本发明培养方法培养了3代的乳腺癌病人的乳腺癌肿瘤细胞进行的肿瘤高频突变分析，HMFL-XN10(P0)是对同一例乳腺癌患者来源的初始分离的未经过传代的乳腺癌肿瘤细胞的分析。上述分析结果使用https://bioinfogp.cnb.csic.es/tools/venny/index.html软件制作。肿瘤高频突变分析结果显示二者的高频突变基因基本一致。HMFL-XN12与HMFL-XN10细胞采用同样的方法平行操作。经图7的(A)确认了本发明技术所培养的癌组织来源的乳腺癌肿瘤细胞能够保持病人癌组织内原始的基因突变特性。
(2)染色体核型分析：采用实施例4的步骤(3)中本发明技术同样的方法将乳腺癌肿瘤细胞(HMFL-XN10)持续培养至第三代(P3)，在收集细胞的前一天在培养基中加入0.1μg/mL的秋水仙胺(Gibico公司制)，16小时后，消化收集细胞并用75mM的低渗透性KCl处理细胞，将细胞用3:1的甲醇︰冰醋酸溶液固定30min。将固定后的细胞均匀地滴加在载玻片上，并使其干燥，再使用5mg/ml的DAPI(Sigma公司制)对细胞核染色15分钟，使用激光共聚焦显微镜(购自Leica公司)拍摄细胞中期分裂相，并对染色体进行配对排列。
图7的(B)显示对HMFL-XN10采用本发明技术培养至第三代的乳腺癌肿瘤细胞的染色体形态和数目正常，没有出现多倍体或低倍体，但核内的染色体复制了两次，且多条染色体彼此靠近，与肿瘤细胞核型特征一致，提示该细胞染色体异常，具有肿瘤细胞特征。由图7的(B)可以确认本发明的培养方法所培养的癌组织来源的乳腺癌肿瘤细胞能够保持乳腺癌病人的染色体核型特性。
[实施例7]
癌组织来源的原代乳腺癌肿瘤细胞在小鼠体内的异种移植成瘤实验
使用与实施例1的步骤(1)同样的方法从两例病理诊断为三阴性乳腺癌患者的癌组织中分离获得乳腺癌肿瘤细胞(HMFL-XN5， HMFL-XN29)，按照实施例2的步骤(2)的方法分别对HMFL-XN5和HMFL-XN29进行培养，待乳腺癌肿瘤细胞数量达到1×10 7个时，采用实施例4的步骤(3)中本发明技术同样的方法对乳腺癌肿瘤细胞进行消化，并收集，采用本发明的乳腺癌肿瘤细胞培养基和Matrigel(注册商标)(BD生物科技公司制)按照1:1混匀，并吸取100μL与Matrigel混匀后的培养基将5×10 6个乳腺癌肿瘤细胞重悬，分别注射入6周大的雌性高度免疫缺陷小鼠(NCG)小鼠(购自南京模式动物研究所)的乳腺脂肪垫和右前肢腋下部位，每三天观察一次乳腺癌肿瘤细胞在小鼠体内形成肿瘤的体积和生长速率，并记录拍照。
由图8可以确认，在肿瘤细胞接种后的第13天即可观察到小鼠的两处肿瘤细胞接种部位均有瘤体形成，自第13天起至第22天，小鼠体内肿瘤增殖明显。说明采用本发明的培养方法所培养的癌组织来源的乳腺癌肿瘤细胞在小鼠体内具有成瘤性。
[实施例8]
癌组织来源的乳腺癌肿瘤细胞的药物敏感性功能测试
下面以乳腺癌患者手术切除样本为例，说明由病人来源的乳腺癌肿瘤样本培养得到的乳腺癌肿瘤细胞可以用于检测病人肿瘤细胞对不同药物的敏感性。
一、原代乳腺癌肿瘤细胞的铺板：将通过实施例4的步骤(3)中本发明技术同样的方法得到的乳腺癌肿瘤细胞(HMFL-XN5、HMFL-XN7、HMFL-XN10、HMFL-XN12和HMFL-XN29)的单细胞悬液，按3000～5000个/孔密度接种于384孔板中，使细胞贴壁过夜。
二、药物梯度实验：
(1)采用浓度梯度稀释的方法配制药物贮存板：分别吸取10μL的待测药物母液(药物母液浓度按2倍于该药物在人体中的最大血药浓度C max配制)，加入到含20μL的DMSO的0.5mL的EP管中，再从上述EP管中吸取10μL到第二个已装有20μL的DMSO的0.5mL的EP管中，即按照1:3稀释药品。重复以上方法，依次稀释，最后得到加药所需的7种浓度。将不同浓度的药物加入384孔药物储存板中。溶剂对照组各孔加入等体积的DMSO作为对照。本实施例中，待测药物为表柔比星(MCE公司制)、拉帕替尼(MCE公司制)、多西他赛(MCE 公司制)和他莫昔芬(MCE公司制)。
(2)使用高通量自动化工作站(购自Perkin Elmer公司)将384孔药物贮存板内的不同浓度药物和溶剂对照加入到铺有乳腺癌肿瘤细胞的384孔细胞培养板中，药物组和溶剂对照组都各设3个复孔。每孔加入药物体积为100nL。
(3)细胞活性检测：给药72小时后，用Cell Titer-Glo检测试剂(Promega公司制)检测加药培养后细胞的化学发光数值，化学发光数值的大小反映细胞活力以及药物对细胞活力的影响，每孔加入配制好的Cell Titer-Glo检测液，混匀后使用酶标仪检测化学发光数值。
使用Graphpad Prism 7.0软件作图并计算半数抑制率IC 50。
(4)药物敏感性测试结果如图9所示。
图9的(A)～(D)分别表示从五个不同的乳腺癌患者的手术切除癌组织样本所培养获得的乳腺癌肿瘤细胞对两个化疗药物表柔比星和多西他赛的药物敏感性、对内分泌治疗药物他莫昔芬的敏感性和对靶向药物拉帕替尼的敏感性。结果显示，同一病人的细胞对不同药物具有不同的敏感性，不同病人的细胞对同一药物的敏感性也不同。
其中，源自激素受体阳性、HER2受体阴性的乳腺癌患者的乳腺癌肿瘤细胞(HMFL-XN7)对内分泌治疗药物他莫昔芬较其他病人敏感，半数抑制率为0.98μM；而对HER2靶向药物拉帕替尼的敏感性较低，半数抑制率为2.5μM。源自HER2阳性乳腺癌患者的乳腺癌肿瘤细胞(HMFL-XN12)的测试结果则显示对抗HER2靶向药物拉帕替尼较其他病人敏感，半数抑制剂量为0.92μM。另外，一例源自三阴性乳腺癌患者的乳腺癌肿瘤细胞(HMFL-XN29)对内分泌治疗药物他莫昔芬和抗HER2靶向药物拉帕替尼的敏感性较其他病人的乳腺癌肿瘤细胞的敏感性偏低。而另一例三阴性乳腺癌患者的乳腺癌肿瘤细胞(HMFL-XN5)则对四种所测试的药物均不敏感。
根据图9确认，本发明技术所培养的乳腺癌病人癌组织来源的乳腺癌肿瘤细胞对化疗药物和靶向药物的敏感性测试结果与病人的临床病理分子分型相符，提示本发明技术所培养的乳腺癌肿瘤细胞在预测乳腺癌患者临床用药疗效方面具有应用潜力。
虽然，上文中已经用一般性说明及具体实施方案对本发明作了详 尽的描述，但在本发明基础上，可以对之作一些修改或改进，这对本领域技术人员而言是显而易见的。因此，在不偏离本发明精神的基础上所做的这些修改或改进，均属于本发明要求保护的范围。
工业上的可利用性
本发明提供一种用于在体外培养或扩增原代乳腺上皮细胞的培养基及培养方法。由本发明的原代乳腺上皮细胞的培养基和培养方法得到的细胞模型，能够用于治疗乳腺疾病的药物的疗效评估或筛选。

Claims (10)

  1. 一种用于培养原代乳腺上皮细胞的原代细胞培养基，其特征在于：
    含有双向调节素。
  2. 如权利要求1所述的原代细胞培养基，其特征在于：
    所述双向调节素的含量为10ng/ml～100ng/ml。
  3. 如权利要求1所述的原代细胞培养基，其特征在于：
    还含有表皮生长因子、胰岛素、B27、ROCK激酶抑制剂Y27632、神经调节素1、成纤维细胞生长因子7、TGFβI型受体抑制剂A8301和P38/MAPK抑制剂SB202190中的一种或多种或全部。
  4. 如权利要求3所述的原代细胞培养基，其特征在于：
    所述表皮生长因子的含量为2.5ng/ml以上，优选为2.5ng/ml～20ng/ml；
    所述胰岛素的含量为1μg/ml～10μg/ml；
    所述B27以1:25～1:100的终浓度稀释；
    所述Y27632含量为5μM～15μM；
    所述神经调节素1的含量为5nM～20nM；
    所述成纤维细胞生长因7的含量为2.5ng/ml～20ng/ml；
    所述A8301的含量为100nM～500nM；
    所述SB202190的含量为100nM～500nM。
  5. 如权利要求1所述的原代细胞培养基，其特征在于：
    不含血清、牛垂体提取物、Wnt激动剂、R-spondin家族蛋白、BMP抑制剂、成纤维细胞生长因子10、烟酰胺和N-乙酰半胱氨酸。
  6. 如权利要求1～5中任一项所述的原代细胞培养基，其特征在于：
    所述原代乳腺上皮细胞为乳腺癌肿瘤细胞、正常乳腺上皮细胞、 乳腺上皮干细胞。
  7. 一种原代乳腺上皮细胞的培养方法，其特征在于，包括以下步骤：
    (1)配制权利要求1～6中任一项所述的原代细胞培养基；
    (2)用细胞外基质胶稀释液包被培养器皿；
    (3)在包被好的培养器皿内接种原代乳腺上皮细胞，使用所述原代细胞培养基，在正常氧气浓度条件或低氧条件下进行培养，在原代乳腺上皮细胞生长至占培养器皿底面积80％～90％左右的细胞密度时进行消化传代。
  8. 如权利要求7所述的培养方法，其特征在于：
    所述细胞外基质胶为低生长因子型，
    采用无血清培养基对所述细胞外基质胶进行稀释，所述细胞外基质胶的稀释比例为1:50～1:400，
    包被方法为将稀释后的细胞外基质胶加入培养器皿内，使其完全覆盖培养器皿底部，静置包被30分钟以上。
  9. 一种评估用于治疗乳腺疾病的药物的疗效的方法，其特征在于，包括以下步骤：
    (1)根据权利要求7或8所述的培养方法培养得到乳腺上皮细胞；
    (2)选定需要检测的药物；
    (3)所述药物以其最大血浆浓度C max为参考，以2～5倍C max为起始浓度，稀释成不同的药物浓度梯度；
    (4)将步骤(1)培养得到的乳腺上皮细胞消化成单细胞悬液，并用含有细胞外基质胶的权利要求1～6中任一项所述的原代细胞培养基将单细胞悬液进行稀释，按每孔1000～10000个细胞的接种密度将稀释后的细胞悬液加入多孔板内，进行过夜贴壁；
    (5)对步骤(4)中得到的贴壁细胞添加梯度稀释后的所述药物；
    (6)进行细胞活力检测。
  10. 如权利要求9所述的方法，其特征在于：
    所述细胞活力检测中，向每孔加入细胞活力检测试剂，震荡均匀后，用荧光酶标仪测量各孔的化学发光强度值，根据测得的数值绘制药物量-效曲线，计算各个药物对细胞的增殖的抑制强度。
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