Culture medium and culture method for lung cancer epithelial cells, and uses thereof

Abstract

A primary cell culture medium for culturing primary lung cancer epithelial cells, comprising at least one additive selected from the group consisting of MST1 / 2 kinase inhibitor, ROCK kinase inhibitor, fibroblast growth factor 7, B27 additive and N2 additive, hepatocyte growth factor, insulin-like growth factor 1, interleukin 6 and TGFβ type I receptor inhibitor. The present invention also relates to a culture method using the primary cell culture medium, and its use in drug efficacy evaluation and drug screening. In the culture method, the primary cell culture medium is used to culture primary cells on a culture vessel coated with an extracellular matrix adhesive so that the primary cells grow. The cell model obtained using the primary cell culture medium and the primary cell culture method can be used for drug efficacy evaluation and drug screening.

Description

[Technical Field]

[0001] The present invention relates to the medical technology field, and in particular to a culture medium and a culture method for culturing or growing primary lung cancer epithelial cells in vitro, as well as a method and use of the cultured cells for evaluating and screening the efficacy of drugs. [Background technology]

[0002] Lung cancer is currently the most common airway tumor worldwide. More than 1.8 million new cases of lung cancer occur worldwide each year. As the most common malignant tumor in clinical practice, lung cancer is primarily treated by surgery, chemotherapy, radiation therapy, molecular targeted therapy, and immunotherapy, with surgery, chemotherapy, and radiation therapy being the most commonly used treatments. However, the populations eligible for these clinical treatments are limited. With recent advances and developments in molecular biology, tumor drug therapies have become increasingly diverse. Molecular targeted drugs, in particular, have become a hot research topic in the clinical treatment of lung cancer due to their powerful targeting and high safety profile. However, among the numerous clinical treatment options, selecting the appropriate one for each patient is particularly important. While genetic testing is used as a guide, determining a treatment plan can be difficult in clinical practice for patients without genetic mutations or for patients with specific mutations for which multiple targeted drugs are available. In addition to gene sequencing, in vitro primary cell culture of samples from lung cancer patients has become an important tool for predicting in vitro efficacy and guiding future clinical medications. However, rapid acquisition of primary lung cancer cells in vitro remains an urgent technical problem to be solved.

[0003] Functional testing refers to an in vitro method for detecting the sensitivity of cancer patient cells to antitumor drugs. The key to applying this method is to develop a tumor cell model with a short growth cycle that can represent the biological characteristics of lung cancer patients. Furthermore, to provide timely and precise drug dosing guidance to cancer patients, this cell model should be easy to manipulate and capable of quickly and efficiently predicting the efficacy of clinical medications. However, the low success rate of establishing cell models in vitro from primary tumor cells of cancer patients, the long growth cycle, and issues such as the overgrowth of mesenchymal cells (e.g., fibroblasts) all limit development in this field. Currently, there are two relatively mature techniques for culturing primary epithelial / stem cells in the field of tumor cell functional testing. One is a technique that uses irradiated feeder cells and the ROCK inhibitor Y27632 to promote the growth of primary epithelial cells and investigate drug sensitivity in individual patients, i.e., conditional cell reprogramming (Non-Patent Document 1). The other technique is to culture adult stem cells in vitro in 3D to obtain organoids that resemble tissues and organs (Non-Patent Document 2).

[0004] However, both technologies have certain limitations. Cell reprogramming is a technique in which autologous primary epithelial cells from a patient are cultured with mouse-derived feeder cells. When testing primary cells from a patient for drug sensitivity, the presence of these mouse-derived cells may interfere with the drug sensitivity test results of the patient's autologous primary cells. However, removing the mouse-derived feeder cells may remove the patient's autologous primary cells from the reprogramming environment, which may significantly alter the cell proliferation rate and intracellular signaling pathways (Non-Patent Document 3, Non-Patent Document 4), thereby significantly affecting the response of the patient's autologous primary cells to drugs. Organoids are a technology in which a patient's autologous primary epithelial cells are embedded in an extracellular matrix for in vitro 3D culture. This technology does not require feeder cells, so there is no problem of interference from mouse-derived feeder cells. However, the culture medium in organoid technology requires the addition of various specific growth factors (e.g., Wnt proteins and R-spondin family proteins), which is expensive and not suitable for widespread clinical use. Furthermore, organoids must be embedded in an extracellular matrix gel throughout the entire culture process. The cell inoculation, passaging, and seeding steps for drug susceptibility testing are laborious and time-consuming compared to 2D culture procedures. Furthermore, it is difficult to control the size of organoids formed by this technique, and some organoids may become too large and develop internal necrosis. Therefore, organoid technology is less maneuverable and applicable than 2D culture techniques. Because it requires specialized technicians to operate, it is not suitable for widespread and widespread use in clinical in vitro drug susceptibility testing (Non-Patent Document 5).

[0005] In light of the limitations of the above techniques, there is a need to develop a culture technique for primary lung cancer epithelial cells in clinical settings that can provide a short culture period, low cost, and easy operation without interference from exogenous cells. By applying this technique to construct primary lung cancer tumor cell models, cultured lung cancer cells can represent the biological characteristics of the lung cancer patient themselves. By evaluating the sensitivity of antitumor drugs in cell models derived from individual cancer patients in vitro, the response rate of antitumor drugs in clinical settings can be improved, reducing the pain caused to patients by inappropriate drugs and the waste of medical resources. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Liu et al., Am J Pathol, 180: 599-607, 2012 [Non-patent document 2] Hans Clevers et al., Cell, 11; 172(1-2): 373-386, 2018 [Non-patent document 3] Liu et al., Am J Pathol, 183(6): 1862-1870, 2013 [Non-patent document 4] Liu et al., Cell Death Dis., 9(7): 750, 2018 [Non-Patent Document 5] Nick Barker, Nat Cell Biol, 18(3): 246-54, 2016 Summary of the Invention

[0007] In view of the shortcomings of the prior art, the present invention aims to provide a culture medium for culturing primary lung cancer epithelial cells and a method for culturing primary lung cancer epithelial cells using the culture medium. The culture medium and culture method for primary lung cancer epithelial cells of the present invention achieve the goals of a short in vitro culture period, cost reduction, and simple operation without interference from exogenous cells. By applying this technology to construct a primary lung cancer tumor cell model, primary lung cancer cells with the biological characteristics of lung cancer patients can be obtained, which can be used in new drug screening and in vitro drug sensitivity testing.

[0008] One aspect of the present invention is to provide a primary cell culture medium for culturing primary lung cancer epithelial cells, comprising an MST1 / 2 kinase inhibitor, at least one ROCK inhibitor selected from the group consisting of Y27632, fasudil, and H-1152, at least one additive selected from the group consisting of fibroblast growth factor 7 (FGF7), B27 additive, and N2 additive, hepatocyte growth factor (HGF), insulin-like growth factor 1 (IGF-1), interleukin 6 (IL-6), and at least one TGFβ type I receptor inhibitor selected from the group consisting of A83-01, SB431542, Repsox, SB505124, SB525334, SD208, LY36494, and SJN2511, wherein the MST1 / 2 kinase inhibitor is represented by formula (I): [ka] (In the formula, R1 is selected from C1-C6 alkyl, C3-C6 cycloalkyl, C4-C8 cycloalkylalkyl, C2-C6 spirocycloalkyl, and aryl optionally substituted with 1-2 independent R6 (e.g., phenyl and naphthyl, etc.), arylC1-C6 alkyl optionally substituted with 1-2 independent R6 (e.g., phenylmethyl, etc.), and heteroaryl optionally substituted with 1-2 independent R6 (e.g., thienyl, etc.); R2 and R3 are each independently selected from C1-C6 alkyl, preferably C1-C3 alkyl, more preferably methyl; R4 and R5 are each independently selected from hydrogen, C1-C6 alkyl, C3-C6 cycloalkyl, C4-C8 cycloalkylalkyl, hydroxylC1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkylaminoC1-C6 alkyl, C1-C6 alkoxyC1-C6 alkyl, and C3-C6 heterocyclylC1-C6 alkyl (heterocyclyl is, for example, selected from piperidyl, tetrahydropyranyl, etc.); R6 includes compounds selected from halogen (preferably fluoro and chloro, more preferably fluoro), C1-C6 alkyl (preferably methyl), C1-C6 alkoxy (preferably methoxy), and C1-C6 haloalkyl (preferably trifluoromethyl), or a pharmaceutically acceptable salt or solvate thereof.

[0009] In a preferred embodiment, the MST1 / 2 kinase inhibitor has formula (Ia): [ka] (In the formula, R1 is selected from C1-C6 alkyl, phenyl optionally substituted with 1-2 independent R6, thienyl optionally substituted with 1-2 independent R6, and phenylmethyl optionally substituted with 1-2 independent R6, more preferably R1 is phenyl optionally substituted with 1-2 independent R6; R5 is selected from hydrogen, C1-C6 alkyl and C3-C6 cycloalkyl, more preferably R5 is hydrogen; R6 is independently selected from halogen, C1-C6 alkyl and C1-C6 haloalkyl; more preferably, R6 is fluoro, methyl or trifluoromethyl), or a pharmaceutically acceptable salt or solvate thereof.

[0010] Preferably, the MST1 / 2 kinase inhibitor is at least one selected from the following compounds or a pharmaceutically acceptable salt or solvate thereof:

[0011] [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5]

[0012] Most preferably, the MST1 / 2 kinase inhibitor of the present invention is Compound 1.

[0013] In an embodiment of the present invention, the amount of the MST1 / 2 kinase inhibitor in the culture medium is generally 2.5 μM to 10 μM, preferably 5 μM to 7.5 μM.

[0014] In yet another embodiment, the ROCK inhibitor is preferably Y27632. Also, preferably, the amount of the ROCK inhibitor in the culture medium is generally 2.5 μM to 15 μM, preferably 5 μM to 15 μM, more preferably 10 μM to 12.5 μM.

[0015] In a preferred embodiment, the amount of fibroblast growth factor 7 is 5 ng / ml to 160 ng / ml, more preferably 10 ng / ml to 80 ng / ml, and even more preferably 20 ng / ml to 40 ng / ml, the volume concentration of the B27 additive or the N2 additive in the culture medium is 1:25 to 1:800, more preferably 1:25 to 1:200, and even more preferably 1:50 to 1:100, the amount of hepatocyte growth factor is 5 ng / ml to 160 ng / ml, and more preferably 20 ng / ml to 80 ng / ml, the amount of insulin-like growth factor 1 is 5 ng / ml to 160 ng / ml, and more preferably 20 ng / ml to 80 ng / ml, the amount of interleukin 6 is 2.5 ng / ml to 40 ng / ml, and more preferably 5 ng / ml to 40 ng / ml, the TGFβ type I receptor inhibitor is preferably A83-01, and the TGFβ The amount of the type I receptor inhibitor is 62.5 nM to 500 nM, preferably 250 nM to 500 nM.

[0016] The medium formulation of the present invention further comprises an initial 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. In some embodiments, the initial medium is preferably DMEM / F12, and the antibiotic is preferably primocin. In further preferred embodiments, the amount of primocin in the culture medium is 25 μg / ml to 400 μg / ml, preferably 50 μg / ml to 200 μg / ml.

[0017] Compared with the composition of the medium for conditional cell reprogramming of lung cancer epithelial cells and the composition of the medium for organoids, the composition of the medium of the present invention contains an MST1 / 2 kinase inhibitor, but does not contain serum, uncertain components such as bovine pituitary extract, niche factors necessary for organoid culture such as Wnt agonists, R-spondin family proteins, and BMP inhibitors, and does not contain nicotinamide or N-acetylcysteine, which significantly reduces the cost of the medium, simplifies the operation process of medium preparation, and enables cost control and easy-to-operate in vitro culture of primary lung cancer epithelial cells.

[0018] In the present invention, the primary lung cancer epithelial cells can be lung cancer cells, normal lung cancer epithelial cells, or lung cancer epithelial stem cells.

[0019] One aspect of the present invention is to provide a method for culturing primary lung cancer epithelial cells, comprising the steps of:

[0020] (1) A step of preparing the primary cell culture medium of the present invention according to the above formulation.

[0021] (2) Coating the culture vessel with a diluted extracellular matrix gel solution.

[0022] Specifically, a low-growth-factor extracellular matrix gel can be used as the extracellular matrix gel, such as commercially available Matrigel (purchased from Corning) or BME (purchased from Trevigen). More specifically, the extracellular matrix gel is diluted with a serum-free culture medium, which may be DMEM / F12 (purchased from Corning). The dilution ratio of the extracellular matrix gel is 1:50 to 1:400, preferably 1:100 to 1:200. The coating method involves adding the diluted extracellular matrix gel to a culture vessel to completely cover the bottom of the culture vessel and leaving it for 30 minutes or more, preferably at 37°C, for a coating time of 30 to 60 minutes. After coating is complete, the excess diluted extracellular matrix gel is discarded, and the culture vessel is ready for subsequent use.

[0023] (3) isolating primary lung cancer epithelial cells from lung cancer tissue.

[0024] Primary lung cancer epithelial cells can be obtained, for example, from lung cancer surgical specimens and biopsy specimens. For example, lung cancer tissue specimens are obtained by surgical resection from lung cancer patients with informed consent, and biopsy specimens are collected from intrapulmonary lesions under ultrasound guidance. The collection of the above-mentioned tissue specimens is performed within 30 minutes of the patient's surgical resection or biopsy. More specifically, in a sterile environment, tissue specimens from non-necrotic sites are cut into 5 mm pieces. 3 After the tissue sample is excised to the above volume, it is placed in 10-15 ml of pre-chilled DMEM / F12 medium, which is then placed in a sterile plastic centrifuge tube with a lid and transported to the laboratory on ice. The DMEM / F12 medium contains an MST1 / 2 kinase inhibitor of the present invention (e.g., Compound 1) and 0.2-0.4 vol% primocin (hereinafter referred to as tissue transport solution). When using an MST1 / 2 kinase inhibitor of the present invention, its concentration range is 0.3-10 μM, preferably 2-5 μM, more preferably 3 μM. When using primocin, its concentration range is 25-400 μg / ml, preferably 50-200 μg / ml, more preferably 100 μg / ml.

[0025] In a biological safety cabinet, transfer the tissue sample to a cell culture dish, rinse it with tissue transport solution, and wash away any blood cells on the surface of the tissue sample. Transfer the rinsed tissue sample to another new culture dish, add 1 to 3 ml of tissue transport solution, and use a sterile scalpel blade and forceps to separate the tissue sample into a volume of 3 mm. 3 Divide into smaller pieces of tissue.

[0026] Transfer the tissue sample to a centrifuge tube and centrifuge it at 1000-3000 rpm for 3-5 minutes in a tabletop centrifuge (Sigma, 3-18K). After discarding the supernatant, add tissue transport solution and tissue digestion solution in a 1:1 ratio (approximately 5 ml of tissue digestion solution per 10 mg of tissue). The tissue digestion solution is prepared by dissolving 1 mg / ml-2 mg / ml collagenase II, 1 mg / ml-2 mg / ml collagenase IV, 50 U / ml-100 U / ml deoxyribonucleic acid I, 0.5 mg / ml-1 mg / ml hyaluronidase, 0.1 mg / ml-0.5 mg / ml calcium chloride, and 5 mg / ml-10 mg / ml bovine serum albumin in HBSS and RPMI-1640 in a 1:1 volume ratio). The samples were then numbered, sealed with sealing film, and digested in a thermostatic shaker (Zhichu Instrument ZQLY-180N) at 37°C and 200-300 rpm. Completion of digestion was determined by hourly observation. If no obvious tissue blocks were found, digestion could be terminated; otherwise, digestion was continued for a period ranging from 4 to 8 hours until sufficient digestion was achieved. After digestion, the undigested tissue blocks were filtered through a cell filter screen (cell screen mesh size, for example, 70 μm). The tissue blocks on the filter screen were rinsed with tissue transport solution, and the remaining cells were placed in a centrifuge tube. The tube was then centrifuged in a tabletop centrifuge at 1000-3000 rpm for 3-5 minutes. After discarding the supernatant, the remaining cell pellet was inspected to determine whether blood cells remained. If blood cells were present, 3-5 ml of blood cell lysis solution (purchased from Sigma) was added, mixed well, and lysed at 4°C for 10-20 minutes, shaking vigorously every 5 minutes. After lysis, the resulting solution was removed and centrifuged at 1000-3000 rpm for 3-5 minutes. After discarding the supernatant, the primary cell culture medium of the present invention was added and the cells were resuspended. The total number of cells was determined by counting using a flow imaging counter (JIMBIO FIL, Jiangsu Jimbio Technology Co., Ltd.).

[0027] (4) inoculating the primary lung cancer epithelial cells isolated in step (3) into a coated culture vessel and culturing them using the primary cell culture medium obtained in step (1).

[0028] More specifically, 2 × 10 primary lung cancer cells were cultured in one well of a multiwell plate. 4 cells / cm 2 ~8×10 4 cells / cm 2 density (e.g., 4×10 4 cells / cm 2 After adding 2-3 ml of primary epithelial cell culture medium, the plate is cultured in a cell incubator at 37°C and 5% CO for 8-16 days. Fresh primary cell culture medium is replaced every 4 days. When the primary lung cancer epithelial cells have grown to a cell density that occupies approximately 80-90% of the bottom surface area of ​​the multi-well plate, they are digested and passaged.

[0029] Compared to conditional cell reprogramming technology, this seeding process does not require the use of feeder cells, eliminating the steps of culturing and irradiating feeder cells. Compared to organoid technology, this process does not require homogeneous mixing of primary cells and matrix gel on ice to form gel droplets, waiting for the gel droplets to solidify, and then adding medium. Therefore, pre-coated culture vessels can be used directly for seeding primary cells. Furthermore, compared to organoid technology, coated culture vessels require only a small amount of diluted extracellular matrix gel, saving the amount of expensive extracellular matrix gel and simplifying the operation process.

[0030] Optionally, after culturing the inoculated primary lung cancer epithelial cells for 8 to 16 days, when the cell clones formed in the culture vessel have converged to cover 80% of the bottom surface, discard the supernatant and add 0.5 to 2 ml of 0.05% trypsin (purchased from Thermo Fisher Scientific) for cell digestion, followed by incubation at room temperature for 5 to 20 minutes. The digested cells are resuspended in 1 to 4 ml of DMEM / F-12 medium containing, for example, 5% (vol / vol) fetal bovine serum, 100 U / ml penicillin, and 100 μg / ml streptomycin, and centrifuged at 1,000 to 3,000 rpm for 3 to 5 minutes. The digested single cells are resuspended using the primary cell culture medium of the present invention, and the resulting cell suspension is placed in a T25 cell culture flask coated with extracellular matrix gel for continuous culture. The coating process for the T25 cell culture flask is the same as in step (2).

[0031] The expanded lung cancer epithelial cells grow in 2D, avoiding unevenly sized organoids and the internal necrosis of overgrown organoids that can occur when expanding using organoid technology.

[0032] In another embodiment, lung cancer epithelial cells, particularly lung cancer cells, cultured by the method for culturing primary lung cancer epithelial cells of the present invention can be used for evaluating and screening the efficacy of drugs, which comprises the following steps:

[0033] (1) Obtaining primary lung cancer epithelial cells, more preferably, obtaining a cancer tissue sample or a biopsy cancer tissue sample derived from a lung cancer patient, isolating the primary lung cancer epithelial cells, and culturing at least 10 primary lung cancer epithelial cells (particularly primary lung cancer cells) according to the method for culturing the primary lung cancer epithelial cells. 5 Size of pieces, preferably at least 10 6 The process of culturing and growing cells until the number of cells reaches the size of a single cell.

[0034] (2) Selecting the drugs to be tested.

[0035] (3) The maximum plasma concentration of the drug as a reference, C maxBased on the initial concentration, C max and diluting the drug into various drug concentration gradients, for example, 5 to 10 drug concentration gradients, preferably 6 to 8 drug concentration gradients.

[0036] (4) digesting the lung cancer epithelial cells cultured in step (1) to prepare a single-cell suspension, counting the number of cells using a flow imaging counter, diluting the single-cell suspension with the primary cell culture medium of the present invention, and uniformly adding the diluted cell suspension to a multi-well plate at a density of 2,000 to 4,000 cells per well, for example, in 50 μl of cell dilution solution per well, and allowing the cells to adhere overnight.

[0037] This step avoids the problem of feeder cells interfering with primary cell counting and subsequent primary cell viability assays in cell reprogramming technology, and eliminates the need for the laborious process of mixing a cell suspension containing a matrix gel on ice, embedding, and then seeding, as in organoid technology, thereby greatly simplifying the procedure and improving the operability and practicality of the technology. Because the cells seeded are single-cell suspensions rather than 3D structures like organoids, this technology allows for more uniform seeding cell numbers and less variation between wells compared to organoid technology, making it more suitable for subsequent high-throughput drug screening procedures.

[0038] (5) Adding selected candidate drugs, such as conventional chemotherapeutic drugs, targeted drugs, antibody drugs, or combinations thereof, in gradient dilutions to the adherent cells obtained in step (4) using a high-throughput automated workstation.

[0039] (6) A few hours after adding the drug, for example, 72 hours after adding the drug, detect the viability of lung cancer epithelial cells using Cell Titer-Glo Luminescent Cell Viability Assay Kit (purchased from Promega) to screen for drug activity.

[0040] Specifically, for example, 10 μl of Cell Titer-Glo reagent (purchased from Promega) is added to each well, and after shaking evenly, the chemiluminescence intensity of each well is measured using a fluorescence microplate reader. Using GraphPad Prism software, a drug dose-effect curve is created based on the measured values, with the drug concentration on the horizontal axis and the fluorescence intensity on the vertical axis, and the inhibitory potency of the drug on the proliferation of the tested cells is calculated.

[0041] When the primary lung cancer cells of the present invention are used for drug screening and in vitro drug sensitivity detection, the lack of a cell co-culture system prevents feeder cells from interfering with the detection results, unlike cell reprogramming technology. Furthermore, due to the 2D cell growth, the drug interaction time is shorter than that of organoid technology (the average administration time in organoid technology is 6 days).

[0042] Beneficial effects of the present invention also include:

[0043] (1) The success rate of primary lung cancer epithelial cell culture is improved with a success rate of over 80%.

[0044] (2) Primary culture of lung cancer epithelial cells in vitro ensures that the pathological phenotype and heterogeneity of the patients from whom the primary cells were derived are recapitulated.

[0045] (3) The primary lung cancer epithelial cells are cultured without interference from fibroblasts, allowing the production of pure lung cancer epithelial cells.

[0046] (4) The medium composition is serum-free, so it is not affected by the quality and quantity of serum from different batches.

[0047] (5) Lung cancer epithelial cells proliferate efficiently, and 10 4 From a starting cell count of 10 cells to 10 cells within approximately 2 weeks 6 Lung cancer epithelial cells were successfully expanded to a scale of 100 cells, and the expanded lung cancer epithelial cells have the ability to be continuously passaged.

[0048] (6) There is no need to operate on ice, there is no need to dissociate the matrix gel during the passaging process, and cell digestion and passaging can be completed within 10 to 15 minutes.

[0049] (7) The primary lung cancer culture medium does not require expensive factors such as Wnt agonists, R-spondin family proteins, or BMP inhibitors, thereby reducing culture costs and simplifying and improving existing culture media for primary lung cancer epithelial cell organoids. Cell seeding does not require the use of highly concentrated extracellular matrix to form gel droplets by mixing with primary cells; instead, a small dilution of the extracellular matrix gel is used, thereby saving the amount of more costly extracellular matrix.

[0050] (8) Simple operation: Compared to conditional reprogramming technology, this technology does not require culturing and irradiating feeder cells, thereby avoiding the problem that the quality and quantity of feeder cells from different batches can affect the efficiency of primary cell culture. In drug screening, only primary lung cancer epithelial cells are seeded and tested, and unlike the co-culture system required in conditional cell reprogramming technology, there is no interference from feeder cells. Compared to organoid technology, the method of coating extracellular matrix gel used in this invention allows the culture vessel to be prepared in advance, and unlike organoid technology, there is no need to embed cells in matrix gel, making the operation process simple and easy.

[0051] (9) This technology allows for the mass cultivation of lung cancer epithelial cells with high uniformity, making it suitable for high-throughput screening of new candidate compounds and for in vitro high-throughput drug sensitivity functional testing in patients.

[0052] The cell culture medium of this embodiment can be used to culture lung cancer epithelial cells derived from humans or other mammals, including lung cancer cells, normal lung epithelial cells, lung cancer epithelial stem cells, or tissues containing at least one of these cells. At the same time, it is also possible to develop a kit for growing and culturing primary lung cancer cells in vitro using the culture medium of the present invention.

[0053] Furthermore, the cells obtained by the culture method of this embodiment can be used in regenerative medicine, basic medical research on lung cancer epithelial cells, screening of drug efficacy, and development of new drugs for lung cancer. [Brief explanation of the drawings]

[0054] [Figure 1-1] 1A to 1C show the effects of different factors in the culture medium on the proliferation of primary lung cancer cells. [Figure 1-2] FIG. 1D shows the effects of different factors in the culture medium on the proliferation of primary lung cancer cells. [Figure 2] FIG. 1 shows the effect of increasing factors in the culture medium on the proliferation of primary lung cancer cells. [Figure 3-1] 3A to 3C are diagrams showing the effect of the concentration of each factor on the proliferation of primary lung cancer cells. [Figure 3-2] 3D to 3F are diagrams showing the effect of the concentration of each factor on the proliferation of primary lung cancer cells. [Figure 3-3] 3G to 3H show the effect of the concentration of each factor on the proliferation of primary lung cancer cells. [Figure 4] Figures 4A and 4B are photographs of lung cancer cells isolated from a single clinical lung cancer tissue sample, cultured for 4 and 12 days, respectively, using the culture medium FLM of the present invention, taken under an inverted microscope. [Figure 5] 5A to 5D are photographs taken under an inverted microscope of cells isolated from a single surgically resected lung cancer sample after culturing them for 12 days under four different culture medium conditions. [Figure 6]This is a comparison of the cell proliferation effects obtained by culturing cells isolated from nine surgically resected lung cancer samples under four different culture medium conditions for 16 days. [Figure 7] 1 is a comparative graph of cell growth curves obtained by culturing cells isolated from one clinical lung cancer tissue sample under four different culture medium conditions. [Figure 8] FIG. 1 shows a comparison of immunohistochemical results of lung cancer cells obtained by culturing cells isolated from one surgically resected lung cancer sample using the culture medium FLM of the present invention. [Figure 9] This is a comparative graph of cell growth curves obtained by culturing cells isolated from a single clinical lung cancer tissue sample in the culture medium FLM of the present invention and in culture media obtained by removing various components from FLM. [Figure 10] 1 is a comparative graph of cell counts after cells isolated from one clinical lung cancer tissue sample were cultured for 8 days in the culture medium FLM of the present invention and in culture media obtained by subtracting various components from FLM. [Figure 11] Figures 11A and 11B show dose-response curves of primary lung cancer cells to different chemotherapeutic drugs and targeted drugs. The primary lung cancer cells were obtained by culturing surgically resected cancer tissue samples from two different lung cancer patients in the FLM culture medium of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0055] As used herein, epithelial cells include differentiated epithelial cells and epithelial stem cells obtained from epithelial tissue. "Epithelial stem cells" are cells that differentiate into epithelial cells and have the ability to self-renew for a long period of time, and are stem cells derived from epithelial tissue. Examples of epithelial tissues include the cornea, oral mucosa, skin, conjunctiva, bladder, renal tubules, kidney, digestive organs (esophagus, stomach, duodenum, small intestine (including jejunum and ileum), large intestine (including colon)), liver, pancreas, mammary gland, salivary gland, lacrimal gland, prostate, hair root, trachea, lung, etc. The cell culture medium of this embodiment is preferably a culture medium for culturing lung-derived epithelial cells.

[0056] Furthermore, as used herein, "epithelial tumor cells" refers to cells obtained by tumorigenesis of cells derived from the above-mentioned epithelial tissues.

[0057] As used herein, "organoid" refers to a three-dimensional organ-like cellular tissue formed by the spontaneous organization and aggregation of cells at high density within a controlled space.

[0058] (Preparation example of MST1 / 2 kinase inhibitor) As used herein, an MST1 / 2 kinase inhibitor refers to any inhibitor that directly or indirectly negatively regulates MST1 / 2 signaling. Generally, an MST1 / 2 kinase inhibitor reduces the activity of MST1 / 2 kinase, for example, by binding to MST1 / 2 kinase. Because MST1 and MST2 have similar structures, an MST1 / 2 kinase inhibitor may be, for example, a compound that binds to MST1 or MST2 and reduces its activity.

[0059] 1. Preparation of MST1 / 2 kinase inhibitor compound 1 4-((7-(2,6-difluorophenyl)-5,8-dimethyl-6-oxo-5,6,7,8-tetrahydropteridin-2-yl)amino)benzsulfamide 1 [ka]

[0060] Methyl 2-amino-2-(2,6-difluorophenyl)acetate (A2): 2-Amino-2-(2,6-difluorophenyl)acetic acid (2.0 g) and methanol (30 ml) were added to a round-bottom flask, followed by the dropwise addition of thionyl chloride (1.2 ml) in an ice bath. The reaction mixture was allowed to react overnight at 85°C. After completion of the reaction, the mixture was evaporated under reduced pressure to dry the solvent, and the resulting white solid was used directly in the next step.

[0061] Methyl 2-((2-chloro-5-nitropyrimidin-4-yl)amino)-2-(2,6-difluorophenyl)acetate (A3): Methyl 2-amino-2-(2,6-difluorophenyl)acetate (2 g) was added to a round-bottom flask, followed by acetone (30 ml) and potassium carbonate (2.2 g). The mixture was then cooled to -10°C in an ice-salt bath, and a solution of 2,4-dichloro-5-nitropyrimidine (3.1 g) in acetone was slowly added. The reaction mixture was stirred overnight at room temperature. After completion of the reaction, the reaction mixture was filtered, and the solvent was removed from the filtrate under reduced pressure. The residue was purified by pressurized silica gel column chromatography to give compound A3. LC / MS: M+H 359.0.

[0062] 2-Chloro-7-(2,6-difluorophenyl)-7,8-dihydropteridin-6(5H)-one (A4): To a round-bottom flask was added methyl 2-((2-chloro-5-nitropyrimidin-4-yl)amino)-2-(2,6-difluorophenyl)acetate (2.5 g), followed by acetic acid (50 ml) and iron powder (3.9 g). The reaction mixture was stirred at 60°C for 2 hours. After completion of the reaction, the reaction mixture was evaporated under reduced pressure to dryness. The resulting mixture was neutralized to alkaline with saturated sodium bicarbonate solution and extracted with ethyl acetate. The organic phase was washed with water and saturated brine and dried over anhydrous sodium sulfate. The organic phase was filtered and evaporated to dryness under reduced pressure to give the crude product. The crude product was washed with diethyl ether to give compound A4. LC / MS: M+H 297.0.

[0063] 2-Chloro-7-(2,6-difluorophenyl)-5,8-dimethyl-7,8-dihydropteridin-6(5H)-one (A5): 2-Chloro-7-(2,6-difluorophenyl)-7,8-dihydropteridin-6(5H)-one (2 g) and N,N-dimethylacetamide (10 ml) were added to a round-bottom flask and cooled to -35°C. Iodomethane (0.9 ml) and sodium hydride (615 mg) were then added, and the reaction mixture was stirred for 2 hours. After completion of the reaction, the reaction mixture was quenched with water and extracted with ethyl acetate. The organic phase was washed with water and saturated brine, respectively, and dried over anhydrous sodium sulfate. The organic phase was filtered and evaporated to dryness under reduced pressure to give the crude product. The crude product was washed with diethyl ether to give compound A5. LC / MS: M+H 325.0.

[0064] 4-((7-(2,6-Difluorophenyl)-5,8-dimethyl-6-oxo-5,6,7,8-tetrahydropteridin-2-yl)amino)benzsulfamide (1): To a round-bottom flask was added 2-chloro-7-(2,6-difluorophenyl)-5,8-dimethyl-7,8-dihydropteridin-6(5H)-one (100 mg), sulfanilamide (53 mg), p-toluenesulfonic acid (53 mg), and sec-butanol (5 ml). The reaction was stirred at 120° C. overnight. After completion of the reaction, the reaction mixture was filtered and washed with methanol and diethyl ether to give compound 1. LC / MS: M+H 461.1.

[0065] 2. Preparation of Other MST1 / 2 Inhibitor Compounds of the Invention Other MST1 / 2 inhibitor compounds of the present invention were synthesized in a similar manner to compound 1, and their structures and mass spectral data are shown in the table below.

[0066] [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5]

[0067] Example 1 Isolation of human primary lung cancer epithelial cells Lung cancer tissue samples were obtained by surgical resection from lung cancer patients who provided informed consent. One of the samples (number B4) is as follows:

[0068] The above-mentioned tissue samples were collected within 30 minutes after surgical resection or biopsy of the patient. More specifically, tissue samples from non-necrotic areas were collected in a sterile environment using 0.5 cm 3 The tissue was excised to the above volume and placed in 4 ml of pre-cooled tissue transport solution (specific formulation shown in Table 1). The transport solution was placed in a 5 ml plastic sterile cryopreservation tube with a lid (purchased from Guangzhou Jet Bio-Filtration Co., Ltd.) and transported to the laboratory via cold chain (0°C to 10°C).

[0069] [Table 3]

[0070] [Table 4]

[0071] In a biological safety cabinet, a tissue sample (number B4) was transferred to a 100 mm cell culture dish (purchased from NEST). The tissue sample was rinsed with tissue transport solution. Residual blood on the surface of the tissue sample was washed away. Excess tissue, such as fat, on the surface of the tissue sample was removed. The rinsed tissue sample was transferred to another new 100 mm culture dish, 2 ml of transport solution was added, and the tissue sample was divided into 3 mm tissues using a sterile scalpel blade and forceps. 3 The tissue was separated into smaller pieces.

[0072] The tissue sample pieces were transferred to a 15 ml centrifuge tube and centrifuged at 1500 rpm for 4 minutes in a tabletop centrifuge (Sigma, 3-18K). After discarding the supernatant, tissue transport solution and tissue digestion solution were added in a 1:1 ratio (the dosage was approximately 5 ml of tissue digestion solution for 10 mg of tissue; the specific formulation is shown in Table 2). The samples were then numbered and sealed with a sealing film before being digested at 37°C and 300 rpm in a thermostatic shaker (Zhichu Instrument ZQLY-180N). Completion of the digestion was determined by hourly observation.

[0073] After digestion, the undigested tissue block was removed by filtration through a 70 μm filter screen, the tissue block on the filter screen was rinsed with tissue transport solution, and the remaining cells were rinsed into a centrifuge tube and centrifuged at 1500 rpm for 4 minutes.

[0074] After discarding the supernatant, the remaining cell pellet was inspected for residual blood cells. If blood cells were present, 3 ml of blood cell lysis solution (purchased from Sigma) was added and mixed thoroughly. The cells were lysed at 4°C for 15 minutes, shaking every 5 minutes. The lysed material was removed and centrifuged at 1500 rpm for 4 minutes. The supernatant was discarded to obtain digested and separated primary lung cancer cells, which were then resuspended in basal medium (BM). Basal medium was prepared by adding 0.2% vol. of primocin (purchased from Invivogen, 50 mg / ml) to commercially available DMEM / F-12 medium to a final concentration of 100 μg / ml. The total cell count was 1,210,000 cells using a flow imaging counter (JIMBIO FIL, Jiangsu Jimbio Technology Co., Ltd.).

[0075] Example 2 Optimization of culture medium for primary lung cancer epithelial cells (1) Influence of different factors An extracellular matrix solution was prepared by diluting extracellular matrix gel (Matrigel™) (BD Biosciences) in serum-free DMEM / F12 medium at a ratio of 1:100. The diluted extracellular matrix solution was added to a 48-well culture plate at 500 μl / well to completely cover the bottom of the wells. The culture plate was left in a 37°C incubator for 1 hour. After 1 hour, the diluted extracellular matrix solution was removed to obtain a Matrigel-coated plate.

[0076] Preparation of basal medium (abbreviated as BM): BM was prepared by adding 0.2% by volume of primocin (purchased from Invivogen, concentration 50 mg / ml) to commercially available DMEM / F-12 medium to give a final concentration of 100 μg / ml.

[0077] Next, different types and concentrations of additive factors (Table 3) were added to the basal medium (BM) to prepare culture media for lung cancer epithelial cells containing various additive components.

[0078] [Table 5]

[0079] Culture media containing various components were added to a 48-well plate coated with extracellular matrix gel (Matrigel) at a volume of 500 μl / well. Lung cancer cells (number B5) isolated from lung cancer tissue according to the same method as described in Example 1 were added to the above-mentioned 48-well culture plate coated with Matrigel at a density of 2 × 10 4 cells / cm 2The plates were seeded at a cell density of 1000 kJ / well. After surface disinfection, the plates were placed in an incubator (purchased from Thermo Fisher) at 37°C and 5% CO2, and the same number of freshly isolated lung cancer tumor cells (number B5) were cultured under different media formulations. The culture medium was changed every four days after the start of culture. After 12 days of culture, cell counts were performed. Basal medium (BM) without any additives was used as a control. The results are shown in Figures 1A–1D. The vertical axis of each figure represents the ratio of the cell number obtained after culture in the different media to the number obtained after culture in basal medium BM. As shown in the figures, the addition of different factors to BM at various concentrations (see Table 3) had different effects on cell proliferation. Among these, B27 additive, N2 additive, fibroblast growth factor 7, interleukin 6, hepatocyte growth factor, insulin-like growth factor 1, compound 1, Y27632, and A83-01 had a certain promoting effect on cell proliferation within a specific concentration range.

[0080] (3) Effect of different growth factors in the culture medium on the proliferation of primary lung cancer cells obtained by the method of the present invention An extracellular matrix diluted solution was prepared by diluting extracellular matrix gel (Matrigel™) (purchased from BD Biosciences) in serum-free DMEM / F12 medium at a ratio of 1:100. 500 μl of the diluted extracellular matrix solution was added to a 48-well culture plate at each well to completely cover the bottom of the wells. The culture plate was then placed in a 37°C incubator for 1 hour. After 1 hour, the diluted extracellular matrix solution was removed to obtain a Matrigel-coated plate.

[0081] Various small molecules, additives, and growth factors (Table 4) were added to the basal medium BM in order to prepare culture media for lung cancer epithelial cells containing various additive components.

[0082] [Table 6]

[0083] Culture media containing various components were added to a 48-well plate coated with extracellular matrix gel (Matrigel) at a volume of 500 μl / well, and BM medium was used as a control. Lung cancer cells (number B5) isolated from lung cancer tissue according to the method described in Example 1 were added to a 48-well plate coated with Matrigel at 2 × 10 4 cells / cm 2 After surface disinfection, the plates were placed in an incubator (purchased from Thermo Fisher) at 37°C and 5% CO2, and the same number of freshly isolated lung cancer tumor cells (number B5) were cultured under different medium formulations. After 10 days of culture, cell counting was performed. The results are shown in Figure 2. As shown in the figure, in this application, we determined that the most suitable culture medium for the culture and growth of primary lung cancer cells was number 8 (hereinafter abbreviated as FLM).

[0084] (4) Effect of different concentrations of additive factors on the proliferation of primary lung cancer cells obtained in this invention An extracellular matrix solution was prepared by diluting extracellular matrix gel (Matrigel™) (BD Biosciences) in serum-free DMEM / F12 medium at a ratio of 1:100. 200 μl of the diluted extracellular matrix solution was added to a 48-well culture plate at each well, completely covering the bottom of the wells. The culture plate was then placed in a 37°C incubator for 1 hour. After 1 hour, the diluted extracellular matrix solution was removed to obtain a Matrigel-coated plate.

[0085] Preparation of the culture medium for primary lung cancer epithelial cells (abbreviated as FLM) of the present invention: fibroblast growth factor 7 (FGF7) at a final concentration of 40 ng / ml, hepatocyte growth factor (HGF) at a final concentration of 40 ng / ml, insulin-like growth factor 1 (IGF-1) at a final concentration of 40 ng / ml, B27 additive at a final volume ratio of 1:50, compound 1 at a final concentration of 5 μM, Y27632 at a final concentration of 10 μM, TGFβ type I inhibitor A83-01 at a final concentration of 500 nM, and interleukin 6 (IL-6) at a final concentration of 20 ng / ml were added to basal medium (BM) to prepare the culture medium for primary lung cancer epithelial cells.

[0086] Lung cancer epithelial cells derived from cancer tissue were isolated from cancer tissue (sample number B6) of a lung cancer patient using the same method as in Example 1. The lung cancer epithelial cells derived from the cancer tissue were then counted using a flow imaging counter (JIMBIO FIL, Jiangsu Jimbio Technology Co., Ltd.) to determine the total cell number. Then, 4 × 10 cells were placed in a 48-well plate coated with Matrigel™ (purchased from BD Biosciences). 4 cells / cm 2 Cells were seeded at a density of 1000 μg / well. Two milliliters of the prepared primary lung cancer epithelial cell culture medium (FLM) was added to a 48-well plate and then cultured in an incubator (purchased from Thermo Fisher Scientific) at 37°C with 5% CO2. When the cells in the culture plate had grown to cover approximately 80% of the bottom area, the culture supernatant was discarded and 500 μl of 0.05% trypsin (purchased from Gibco) was added for cell digestion. The plate was then incubated at 37°C for 10 minutes. Microscopic observation (EVOS M500, Invitrogen) confirmed that the cells were completely digested. The digestion was then terminated with 1 ml of DMEM / F12 medium containing 5% (vol / vol) fetal bovine serum (purchased from ExCell Bio), 100 U / ml penicillin (purchased from Corning), and 100 μg / ml streptomycin (purchased from Corning). The resulting material was collected in a 15 ml centrifuge tube and centrifuged at 1500 rpm for 4 minutes, after which the supernatant was discarded. The centrifuged cell pellet was resuspended in basal medium BM and counted using a flow imaging counter (JIMBIO FIL, Jiangsu Jimbio Technology Co., Ltd.) to determine the total cell number. The resulting cells were used in the following culture experiments.

[0087] Next, the following eight types of medium formulations were prepared and experiments were carried out.

[0088] Formulation 1: FLM medium composition without B27 additive; Formulation 2: FLM medium composition without fibroblast growth factor 7; Formulation 3: FLM medium composition without insulin-like growth factor 1; Formulation 4: FLM medium composition without hepatocyte growth factor; Formulation 5: FLM medium composition without Y27632; Formulation 6: FLM medium composition without Compound 1; Formulation 7: FLM medium composition without A83-01; Formulation 8: FLM medium composition without interleukin-6.

[0089] The digested cell suspension was diluted with the above-mentioned formulations 1 to 8, respectively, and then inoculated into a 48-well plate in a volume of 250 μl containing 10,000 cells per well.

[0090] When using the medium of Formulation 1, 250 μl of B27 additive was added per well to the 48-well plate inoculated with primary cells at final concentrations of 1:800, 1:400, 1:200, 1:100, 1:50, and 1:25. Blank control (BC) wells were set up using the medium of Formulation 1.

[0091] When using the medium of Formulation 2, 250 μl of fibroblast growth factor 7 adjusted to final concentrations of 160 ng / ml, 80 ng / ml, 40 ng / ml, 20 ng / ml, 10 ng / ml, or 5 ng / ml was added per well to a 48-well plate inoculated with primary cells. Blank control (BC) wells were set up using the medium of Formulation 2.

[0092] When using the medium of Formulation 3, 250 μl of insulin-like growth factor 1 prepared to the final concentrations of 160 ng / ml, 80 ng / ml, 40 ng / ml, 20 ng / ml, 10 ng / ml, and 5 ng / ml was added per well to the 48-well plate inoculated with primary cells. Blank control (BC) wells were set up using the medium of Formulation 3.

[0093] When using the medium of Formulation 4, 250 μl of hepatocyte growth factor was added per well to the 48-well plate inoculated with primary cells to final concentrations of 160 ng / ml, 80 ng / ml, 40 ng / ml, 20 ng / ml, 10 ng / ml, and 5 ng / ml, respectively. Blank control (BC) wells were set up using the medium of Formulation 4.

[0094] When using the medium of Formulation 5, 250 μl of Y27632 was added per well to a 48-well plate inoculated with primary cells to final concentrations of 20 μM, 15 μM, 12.5 μM, 10 μM, 5 μM, and 2.5 μM. Blank control (BC) wells were set up using the medium of Formulation 5.

[0095] When using the medium of Formulation 6, Compound 1 was added to a 48-well plate inoculated with primary cells at final concentrations of 20 μM, 10 μM, 7.5 μM, 5 μM, 2.5 μM, and 1.25 μM in an amount of 250 μL per well. Blank control (BC) wells were set up using the medium of Formulation 6.

[0096] When using the medium of Formulation 7, 250 μl of A83-01 was added per well to the 48-well plate inoculated with primary cells to final concentrations of 2000 nM, 1000 nM, 500 nM, 250 nM, 125 nM, and 62.5 nM. Blank control (BC) wells were set up using the medium of Formulation 7.

[0097] When using the medium of Formulation 8, 250 μl of interleukin-6 prepared to the final concentrations of 80 ng / ml, 40 ng / ml, 20 ng / ml, 10 ng / ml, 5 ng / ml, and 2.5 ng / ml was added per well to the 48-well plate inoculated with primary cells. Blank control (BC) wells were set up using the medium of Formulation 8.

[0098] After cells were grown to approximately 85% of the 48 wells, they were digested and counted. The ratios were calculated based on the cell counts in the blank control (BC) wells. The results are shown in Figures 3A-3H. In each of Figures 3A-3H, the ratios represent the ratio of the number of cells at the first passage cultured using each culture medium to the number of cells at the first passage cultured in the corresponding blank control well. A ratio greater than 1 indicates that the growth-promoting effect of the conditioned medium containing different concentrations of factors or small molecule compounds is more favorable than that of the medium in the blank control wells. A ratio less than 1 indicates that the growth-promoting effect of the conditioned medium containing different concentrations of factors or small molecule compounds is worse than that of the medium in the blank control wells.

[0099] According to the results of Figures 3A to 3H, the volume concentration of B27 additive in the culture medium is preferably 1:25 to 1:800, more preferably 1:25 to 1:200, and even more preferably 1:50 to 1:100, the amount of fibroblast growth factor 7 is preferably 5 ng / ml to 160 ng / ml, more preferably 10 ng / ml to 80 ng / ml, and even more preferably 20 ng / ml to 40 ng / ml, the amount of insulin-like growth factor 1 is preferably 5 ng / ml to 160 ng / ml, more preferably 20 ng / ml to 80 ng / ml, and the amount of hepatocyte growth factor is preferably 5 ng / ml to 160 ng / ml, more preferably 20 ng / ml to 80 ng / ml. The amount of Y27632 is preferably 2.5 μM to 15 μM, more preferably 5 μM to 15 μM, and even more preferably 10 μM to 12.5 μM; the amount of Compound 1 is preferably 2.5 μM to 10 μM, more preferably 5 μM to 7.5 μM; the amount of A83-01 is preferably 62.5 nM to 500 nM, more preferably 250 nM to 500 nM; and the amount of interleukin 6 is preferably 2.5 ng / ml to 40 ng / ml, more preferably 5 ng / ml to 40 ng / ml.

[0100] Example 3 Cultivation of primary lung cancer cells derived from lung cancer tissue Lung cancer epithelial cells derived from cancer tissue were isolated from cancer tissue (sample number B7) of a lung cancer patient using the same method as in Example 1. The lung cancer epithelial cells derived from the cancer tissue were then counted using a flow imaging counter (JIMBIO FIL, Jiangsu Jimbio Technology Co., Ltd.) to determine the total cell number. Then, 4 × 10 cells were placed in a 12-well plate coated with Matrigel™ (purchased from BD Biosciences). 4 cells / cm 2 The cells were seeded at a density of 1000 x g / well. 2 ml of the prepared FLM culture medium for primary lung cancer epithelial cells was added to a 12-well plate, which was then placed in an incubator (purchased from Thermo Fisher) at 37°C and 5% CO2 for culture.

[0101] Figure 4A shows 4 x 10 cells grown in a 12-well plate coated with Matrigel according to this example. 4 cells / cm 2 Figure 4A and Figure 4B show microscopic images (taken with a 40x inverted phase-contrast microscope) of cells cultured at a density of 100 μg / ml for 4 days after seeding. Microscopic observation indicates that the primary lung cancer cells derived from cancer tissue were highly pure and did not contain fibroblasts. Figure 4B shows microscopic images (taken with a 40x inverted phase-contrast microscope) of cells cultured according to this example for 12 days after seeding. Figures 4A and 4B show that when the isolated primary lung cancer cells were cultured in vitro for 4 days, clear clone formation was observed under a microscope, and that the cell number was significantly expanded after 12 days of growth. This suggests that the technique of the present invention is an efficient technique for growing lung cancer epithelial cells in vitro.

[0102] Example 4 Effects of various culture media on the growth of primary lung cancer cells derived from lung cancer tissues (1) Comparison of the effects of various culture media on the clonal formation of first-generation primary cells and their proliferation effects A culture medium for primary lung cancer epithelial cells (FLM) was prepared in the same manner as in Example 2, and a basal medium (BM) was prepared as a control. As another control, a culture medium (FM) for use in conditional cell reprogramming technology was additionally prepared. For the preparation process, see Liu et al., Nat Protoc., 12(2):439-451, 2017. The formulation of the culture medium is shown in Table 5. Furthermore, as an additional control, a commercially available medium, EpiMCult™ Plus Medium (hereinafter also referred to as "EpiM medium"), was purchased from STEMCELL, and the formulation of the culture medium is shown in Table 6.

[0103] [Table 7]

[0104] [Table 8]

[0105] Primary lung cancer cells (number B8) derived from lung cancer tissue were obtained using the same method as in Example 1. Next, cells were cultured at the same density (4 × 10) under the following five culture conditions. 4 cells / cm 2 ) and cultured the cells.

[0106] A. Technique of the present invention: 4×10 primary lung cancer cells were plated on a 24-well plate coated with Matrigel™ (purchased from BD Biosciences). 4 cells / cm 2 The cells were seeded at a density of 1000×1000×1000 and cultured in 2 ml of the primary lung cancer epithelial cell culture medium FLM of the present invention.

[0107] B. Conditional cell reprogramming technique: γ-irradiated mouse fibroblast line J2 cells (purchased from Kerafast) were pre-arranged in a 24-well plate with 4 × 10 primary lung cancer cells. 4 cells / cm 2The cells were seeded at a density of 1000×1000×1000 and cultured in a 24-well plate using conditioned cell reprogramming medium FM (for detailed steps, see Non-Patent Document 3).

[0108] C. 4 × 10 primary lung cancer cells were plated on a 24-well plate coated with Matrigel™ (BD Biosciences). 4 cells / cm 2 The cells were inoculated at a density of 1000 kJ / well and cultured in 2 ml of the commercially available culture medium EpiM in a 24-well plate.

[0109] D. 4 × 10 primary lung cancer cells were plated on a 24-well plate coated with Matrigel™ (BD Biosciences). 4 cells / cm 2 The cells were inoculated at a density of 1000×1000 and cultured in 2 ml of basal medium BM in a 24-well plate.

[0110] The cells were cultured under the four culture conditions, with the medium renewed every four days. Cell clone formation and cell proliferation were observed in 24-well plates and recorded by photographing the cell growth under a microscope (EVOS M500, Invitrogen).

[0111] For primary lung cancer cells (number B8) cultured using the present technology, when the cells in the culture plate grew to cover approximately 80% of the bottom area, the culture supernatant in the 24-well plate was discarded, and 500 μl of 0.05% trypsin (purchased from GIBCO) was added for cell digestion. The cells were then incubated at 37°C for 10 minutes. Microscopic observation (EVOS M500, Invitrogen) revealed complete cell digestion. The digestion was then terminated with 1 ml of DMEM / F12 medium containing 5% (vol / vol) fetal bovine serum, 100 U / ml penicillin, and 100 μg / ml streptomycin. The resulting material was collected in a 15 ml centrifuge tube and centrifuged at 1500 rpm for 4 minutes, after which the supernatant was discarded. The centrifuged cell pellet was resuspended in the culture medium of the present invention, and the cells were counted using a flow imaging counter (JIMBIO FIL, Jiangsu Jimbio Technology Co., Ltd.) to determine the total cell number, which was 464,000. Cells cultured under the other three culture conditions were digested and counted using the same procedure as above. The total cell numbers cultured using FM, EpiM, and BM media were 350,000, 110,000, and 68,000, respectively.

[0112] Figures 5A-5D are micrographs (under a 40x inverted phase-contrast microscope) of sample number B8, which was cultured for 12 days under four different culture conditions. Figure 5A is a micrograph of B8 cultured for 12 days using basal medium BM. Figure 5B is a micrograph of B8 cultured for 12 days using the culture medium FLM of the present invention. Figure 5C is a micrograph of B8 cultured for 12 days using the commercially available medium EpiM. Figure 5D is a micrograph of B8 cultured for 12 days using conditional reprogramming medium FM. As can be seen from the figures, sample B8 failed to form cell clones even after 12 days of culture in basal medium BM (Figure 5A). Even after 12 days of culture in EpiM (Figure 5C), only a few cell clones were formed, resulting in poor cell condition. When cultured for 12 days in conditional reprogramming medium FM (Figure 5D), cells proliferated to some extent, but the cell density and cell number were incomparable to those in the medium FLM of the present invention.

[0113] Figure 6 shows a comparison of the cell proliferation effects obtained by culturing primary lung cancer cells isolated from nine lung cancer patient samples in accordance with Example 1 under four different culture medium conditions for 16 days, where √ indicates moderate clonogenicity and proliferation-promoting effect, √√ indicates significant clonogenicity and proliferation-promoting effect, √√√ indicates highly significant clonogenicity and proliferation-promoting effect, and × indicates no clonogenesis. Figure 6 confirms that the culture medium of the present invention is significantly superior to the other three culture conditions in terms of clonogenicity, cell proliferation-promoting effect, and culture success rate when culturing primary cells derived from lung cancer tissues.

[0114] (2) Continuous culture and growth curve of primary lung cancer cells in various culture media Using the same method as in (1) of this Example, a culture medium FLM for primary lung cancer epithelial cells and control culture media BM, FM, and EpiM were obtained.

[0115] Primary lung cancer cells (number B9) derived from lung cancer tissue were cultured under four different culture conditions using the same method as in (1) of this Example, and then digested, passaged, and counted.

[0116] When the passaged cells had grown again in the culture plate to cover approximately 80% of the bottom surface area of ​​the plate, the cultured cells were digested, collected, and counted according to the procedure described above. The cells were again collected at 4 × 10 4 The cells were seeded at a density of 100 cells / well and cultured continuously.

[0117] Below is the formula for calculating the population doublings of primary lung cancer epithelial cells under different culture conditions. Population doubling (PD) = 3.32 × log 10 (total number of digested cells / number of cells at initial inoculation), see Chapman et al., Stem Cell Research & Therapy 2014, 5:60.

[0118] Figure 7 shows the growth curves of B9 cells under four different culture conditions plotted using Graphpad Prism software. The horizontal axis represents the number of days in cell culture, and the vertical axis represents the cumulative cell proliferation fold, i.e., the fold increase in cell proliferation during the culture period. The higher the value, the more times the cells have proliferated within a given period of time, i.e., the more cells have proliferated. The slope represents the cell proliferation rate. The figure confirms that the proliferation rate of lung cancer epithelial cells cultured in the culture medium FLM of the present invention was superior to that of the other four culture conditions. It also confirms that the culture medium of the present invention allows continuous culture of primary lung cancer epithelial cells, with no change in proliferation rate even up to 30 days.

[0119] Example 5 Immunohistochemical identification of lung cancer cells in primary and subcultured lung cancer tissues A cancer tissue (sample number B12) approximately the size of a soybean was collected from a surgically resected specimen of a lung cancer patient and immersed in 1 ml of 4% paraformaldehyde. Lung cancer epithelial cells (sample number B12) were obtained from the remaining cancer tissue using the same method as in Example 1. Sample B12 was continuously cultured up to the fourth passage using the culture medium FLM of the present invention according to the method in Example 3.

[0120] Immunohistochemical assays were used to detect the expression of key lung cancer-related biomarkers in the original tissue of sample B12 and in primary cells obtained by continuous culture up to the fourth passage. The tissue was fixed in 4% paraformaldehyde, embedded in paraffin, and then cut into 4-μm-thick tissue sections using a microtome. Standard immunohistochemical detection was then performed (for detailed procedures, see Li et al., Nature Communication, (2018) 9:2983). The primary antibodies used were TTF-1 (purchased from CST) and Ki67 (purchased from R&D).

[0121] 8 confirms that the expression of lung cancer-related biomarkers on cells cultured up to the fourth passage from lung cancer cells (sample number B12) using the culture medium of the present invention was substantially consistent with the expression of markers on the tissue sections from which the cells were derived, suggesting that the cells cultured using the culture medium of the present invention maintain the original pathological characteristics of the cancer tissues of lung cancer patients.

[0122] Example 6 Effects of removal of single factors from culture medium on continuous growth and culture of primary lung cancer cells. A culture medium for primary lung cancer epithelial cells, FLM, was prepared using the same method as in Example 2. As a control, a basal medium, BM, was prepared using the same method as in Example 2. Eight other different culture media were also prepared according to Table 7.

[0123] [Table 9]

[0124] One case of primary lung cancer cells (number B13) derived from lung cancer tissue was obtained using the same method as in Example 1. 4 × 10 primary lung cancer cells were cultured in a 48-well plate coated with Matrigel™ (BD Biosciences). 4 cells / cm 2 The cells were seeded at a density of 1000 x g and cultured in 2 ml of the culture medium for primary lung cancer epithelial cells (FLM) of the present invention in an incubator (purchased from Thermo Fisher) at 37°C and 5% CO2.

[0125] When the cells in the culture plate had grown to cover approximately 80% of the bottom area, the culture supernatant in the 48-well plate was discarded, and 200 μl of 0.05% trypsin (purchased from Gibco) was added for cell digestion. The plate was then incubated at 37°C for 10 minutes, and observation under a microscope (EVOS M500, Invitrogen) confirmed that the cells were completely digested. The digestion was then terminated with 800 μl of DMEM / F12 medium containing 5% (vol / vol) fetal bovine serum, 100 U / ml penicillin, and 100 μg / ml streptomycin. The resulting mixture was collected in a 15 ml centrifuge tube and centrifuged at 1500 rpm for 4 minutes, after which the supernatant was discarded. The centrifuged cell pellet was resuspended in the culture medium of the present invention, and the total cell number was determined by counting the cells using a flow imaging counter (JIMBIO FIL, Jiangsu Jimbio Technology Co., Ltd.). 2 x 10 cells in a separate 48-well plate coated with extracellular matrix gel 4 cells / cm 2 The cells were inoculated at a density of 1000 μg / ml and further cultured.

[0126] Cells cultured in the other eight culture media and BM were digested, passaged, and counted using the same methods as above, and cultured in different media.

[0127] When the passaged cells had grown again in the culture plate to cover approximately 80% of the bottom surface area of ​​the plate, the cultured cells were digested, collected, and counted according to the procedure described above. The cells were again collected at 2 × 10 4 cells / cm 2 The culture was inoculated at a density of 1000 kJ / ml and continuous culture was carried out.

[0128] Below is the formula for calculating the population doublings of primary lung cancer epithelial cells under different culture medium conditions. Population doubling (PD) = 3.32 × log 10 (total number of digested cells / number of cells at initial inoculation), see Chapman et al., Stem Cell Research & Therapy 2014, 5:60.

[0129] Figure 9 shows a graph of cell growth curves under 10 different culture medium conditions, plotted using Graphpad Prism software. The horizontal axis represents the number of days in cell culture, and the vertical axis represents the cumulative cell proliferation fold, i.e., the fold increase in cell proliferation over the culture period. The higher the value, the more times the cells have proliferated within a given time period, i.e., the more cells have proliferated. The slope represents the cell proliferation rate.

[0130] FIG. 10 shows the results of cell harvest and counting after 8 days of culture under 10 different culture medium conditions.

[0131] As can be seen from the results of Figures 9 and 10, when various small molecules and additive factors were removed from the culture medium of the present invention, the cell proliferation effect was weakened to some extent, suggesting that these components in the culture medium of the present invention are necessary for cell proliferation and continuous culture.

[0132] Example 7 Tumorigenesis experiments using xenografts of primary lung cancer cells derived from cancer tissue in mice Lung cancer cells (number B15) were isolated and obtained from cancer tissue of one lung cancer patient who had been pathologically diagnosed, using the same method as in Example 1. B15 was cultured using the culture medium FLM of the present invention according to the method of Example 3, and the number of lung cancer cells reached 1 × 10 7 When the lung cancer cells reached 100 μl, they were digested and collected using the same method as in Example 4. The lung cancer cell culture medium FLM of the present invention and Matrigel™ (purchased from BD Biosciences) were mixed at a 1:1 ratio, and 5 × 10 cells were cultured using 100 μl of the culture medium mixed with Matrigel. 6 The lung cancer cells were resuspended and injected into the lung fat pad and right forelimb axilla of 6-week-old female severely immunodeficient mice (NCG) (purchased from the Nanjing Model Animal Research Center). The tumor volume and growth rate in the mice were monitored and photographed every 3 days.

[0133] Tumor formation was observed at both tumor cell inoculation sites in the mice 15 days after tumor cell inoculation. Tumor growth in the mice was evident from days 15 to 30. This indicates that lung cancer cells derived from cancer tissues cultured by the culture method of the present invention have tumorigenicity in mice.

[0134] Example 8 Functional drug sensitivity testing of lung cancer cells derived from cancer tissue Taking a surgically resected sample from a lung cancer patient as an example, it is confirmed that lung cancer cells cultured from the patient-derived lung cancer sample can be used to test the sensitivity of the patient's tumor cells to various drugs.

[0135] 1. Seeding of primary lung cancer cells: A cell suspension of isolated lung cancer cells (No. B13 and No. B15) obtained according to the method of Example 1 was seeded at 4 × 10 cells / well onto a 12-well plate coated with Matrigel™ (purchased from BD Biosciences). 4 cells / cm 2Cells were seeded at a density of 1000 kJ / well. Two milliliters of prepared primary lung cancer epithelial cell culture medium (FLM) was added to a 12-well plate and then cultured in a 37°C, 5% CO2 incubator (purchased from Thermo Fisher Scientific). When the cells had grown to cover approximately 80% of the bottom area, the culture supernatant was discarded and 500 μl of 0.05% trypsin (purchased from Gibco) was added for cell digestion. The cells were incubated at 37°C for 10 minutes and observed under a microscope (EVOS M500, Invitrogen). The cells were then completely digested. The digestion was then terminated with 1 ml of DMEM / F12 medium containing 5% (vol / vol) fetal bovine serum, 100 U / ml penicillin, and 100 μg / ml streptomycin. The resulting mixture was collected in a 15 ml centrifuge tube and centrifuged at 1500 rpm for 4 minutes, after which the supernatant was discarded. The centrifuged cell pellet was resuspended in FLM culture medium and counted using a flow imaging counter (JIMBIO FIL, Jiangsu Jimbio Technology Co., Ltd.) to determine the total cell numbers, which were 830,000 and 768,000, respectively. Cells were seeded into 384-well plates at densities of 2,000–4,000 cells / well and allowed to adhere overnight.

[0136] 2. Drug gradient experiments: (1) Drug storage plates were prepared by gradient dilution: 10 μl of the drug stock solution to be tested (the drug stock solution is the maximum plasma concentration of the drug in the human body, C maxEach drug (prepared to have a concentration twice that of the original drug) was pipetted into a 0.5 ml EP tube containing 20 μl of DMSO, and 10 μl of the solution from the EP tube was pipetted into a second 0.5 ml EP tube containing 20 μl of DMSO. This means that the drug was diluted at a 1:3 ratio. The above procedure was repeated to serially dilute the drug to obtain the eight concentrations required for dosing. Different concentrations of the drug were added to a 384-well drug storage plate. An equal volume of DMSO was added to each well of the solvent control group as a control. In this example, the drugs tested were paclitaxel (purchased from MCE), gemcitabine (purchased from MCE), afatinib (purchased from MCE), and erlotinib (purchased from MCE).

[0137] (2) Using a high-throughput automated workstation (JANUS, Perkin Elmer), various concentrations of drugs and solvent controls in a 384-well drug reservoir plate were added to a 384-well cell culture plate seeded with lung cancer cells. Each drug group and solvent control group was arranged in triplicate wells. The volume of drug added to each well was 100 nL.

[0138] (3) Cell viability test: 72 hours after administration, the chemiluminescence value of the cultured cells after drug administration was detected using a Cell Titer-Glo assay kit (purchased from Promega). The magnitude of the chemiluminescence value reflects the cell viability and the effect of the drug on cell viability. 10 μl of the prepared Cell Titer-Glo detection solution was added to each well, and the chemiluminescence value was detected after mixing using a microplate reader (Envision, Perkin Elmer).

[0139] (4) Cell viability test: Cell viability (%) = chemiluminescence value of drug wells / chemiluminescence value of control wells × 100%. The cell viability of cells treated with various drugs was calculated using the formula: Cell viability (%) = chemiluminescence value of drug wells / chemiluminescence value of control wells × 100%. Graphs were created using Graphpad Prism software, and the median inhibition rate (IC) was calculated. 50 was calculated.

[0140] (5) The results of the drug susceptibility test are shown in Figure 11.

[0141] Figures 11A and 11B show the sensitivity of lung cancer cells cultured from surgically resected cancer tissue samples (sample numbers B13 and B15) from two different lung cancer patients to two chemotherapeutic drugs, paclitaxel and gemcitabine, and two targeted drugs, afatinib and erlotinib, respectively. Specifically, Figure 11A shows the sensitivity results of lung cancer cells cultured from sample number B13 to four drugs, and Figure 11B shows the sensitivity results of lung cancer cells cultured from sample number B15 to four drugs. These results indicate that cells from the same patient have different sensitivities to different drugs, and cells from different patients also have different sensitivities to the same drug.

[0142] Although the present invention has been described in detail by the above general description and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made based on this specification. Therefore, these modifications or improvements that do not deviate from the spirit of the present invention are included in the protection scope claimed by the present invention. [Industrial Applicability]

[0143] The present invention provides a culture medium and a culture method for culturing or growing primary lung cancer epithelial cells in vitro. These cultured cells can be used for drug efficacy evaluation and screening. Therefore, the present invention is applicable to industry.

[0144] Although the present invention has been described in detail above, the present invention is not limited thereto, and those skilled in the art can make modifications according to the principles of the present invention. Therefore, all modifications made according to the principles of the present invention should be construed as falling within the protection scope of the present invention.

Claims

1. 1. A primary cell culture medium for culturing primary lung cancer epithelial cells, comprising: the composition comprises an MST1 / 2 kinase inhibitor, at least one ROCK inhibitor selected from the group consisting of Y27632, fasudil, and H-1152, at least one additive selected from the group consisting of fibroblast growth factor 7, B27 additive, and N2 additive, hepatocyte growth factor, insulin-like growth factor 1, interleukin 6, and at least one TGFβ type I receptor inhibitor selected from the group consisting of A83-01, SB431542, Repsox, SB505124, SB525334, SD208, LY36494, and SJN2511; The MST1 / 2 kinase inhibitor is represented by formula (I): 【Chemistry 1】 (In the formula, R １ is C3-C6 cycloalkyl, C4-C8 cycloalkylalkyl, C2-C6 spirocycloalkyl, and one to two independent R ６ aryl optionally substituted with one to two independent R ６ aryl C1-C6 alkyl optionally substituted with, and one to two independent R ６ heteroaryl optionally substituted with R ２ and R ３ are each independently selected from C1-C6 alkyl; R ４ and R ５ are each independently selected from hydrogen, C1-C6 alkyl, C3-C6 cycloalkyl, C4-C8 cycloalkylalkyl, hydroxylC1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkylaminoC1-C6 alkyl, C1-C6 alkoxyC1-C6 alkyl, and C3-C6 heterocyclylC1-C6 alkyl; R ６ is selected from halogen, C1-C6 alkyl, C1-C6 alkoxy, and C1-C6 haloalkyl, or a pharmaceutically acceptable salt or solvate thereof.

2. R １ is C3-C6 cycloalkyl, C4-C8 cycloalkylalkyl, C2-C6 spirocycloalkyl, and one to two independent R ６ phenyl optionally substituted with one to two independent R ６ naphthyl optionally substituted with one to two independent R ６ phenylmethyl optionally substituted with, and one to two independent R ６ thienyl optionally substituted with R ２ and R ３ are each independently selected from C1-C3 alkyl; R ４ and R ５ are each independently selected from hydrogen, C1-C6 alkyl, C3-C6 cycloalkyl, C4-C8 cycloalkylalkyl, hydroxylC1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkylaminoC1-C6 alkyl, C1-C6 alkoxyC1-C6 alkyl, piperidylC1-C6 alkyl, and tetrahydropyranylC1-C6 alkyl; R ６ is selected from halogen, C1-C6 alkyl, C1-C6 alkoxy, and C1-C6 haloalkyl; 2. The primary cell culture medium of claim 1.

3. The MST1 / 2 kinase inhibitor is represented by formula (Ia): 【Chemistry 2】 (In the formula, R １ is one to two independent R ６ phenyl optionally substituted with one to two independent R ６ thienyl optionally substituted with, and one to two independent R ６ phenylmethyl optionally substituted with R ５ is selected from hydrogen, C1-C6 alkyl, and C3-C6 cycloalkyl; R ６ are independently selected from halogen, C1-C6 alkyl, and C1-C6 haloalkyl, or a pharmaceutically acceptable salt or solvate thereof.

4. R １ There are one to two independent R ６ is phenyl optionally substituted with R ５ is hydrogen, R ６ 4. The primary cell culture medium of claim 3, wherein is fluoro, methyl, or trifluoromethyl.

5. 2. The primary cell culture medium according to claim 1, wherein the MST1 / 2 kinase inhibitor is at least one selected from the following compounds or pharmaceutically acceptable salts thereof: Table 1-1 Table 1-2 Table 1-3 Table 1-4 Table 1-5

6. 6. The primary cell culture medium according to claim 1, wherein the amount of the MST1 / 2 kinase inhibitor in the culture medium is between 2.5 μM and 10 μM.

7. The following conditions: the amount of the ROCK inhibitor in the culture medium is 2.5 μM to 15 μM; the amount of fibroblast growth factor 7 is between 5 ng / ml and 160 ng / ml; The volume concentration of B27 additive or N2 additive in the culture medium is 1:25 to 1:800; the amount of hepatocyte growth factor is 5 ng / ml to 160 ng / ml; the amount of insulin-like growth factor 1 is between 5 ng / ml and 160 ng / ml; the amount of interleukin 6 is between 2.5 ng / ml and 40 ng / ml; the amount of the TGFβ type I receptor inhibitor is 62.5 nM to 500 nM; The primary cell culture medium according to any one of claims 1 to 5, characterized in that it satisfies one or more or all of the following requirements:

8. The following conditions: the MST1 / 2 kinase inhibitor is Compound 1; the ROCK inhibitor is Y27632; The TGFβ type I receptor inhibitor is A83-01. The primary cell culture medium according to any one of claims 1 to 5, characterized in that it satisfies one or more or all of the following requirements:

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

10. The primary cell culture medium according to any one of claims 1 to 5, characterized in that it does not contain serum, bovine pituitary extract, Wnt agonist, R-spondin family protein, BMP inhibitor, nicotinamide, or N-acetylcysteine.

11. The primary cell culture medium according to any one of claims 1 to 5, wherein the primary lung cancer epithelial cells comprise lung cancer epithelial stem cells.

12. 1. A method for culturing primary lung cancer epithelial cells, comprising: (1) preparing a primary cell culture medium according to any one of claims 1 to 11; (2) coating the culture vessel with a diluted extracellular matrix gel selected from at least one of Matrigel and BME; (3) inoculating primary lung cancer epithelial cells isolated from lung cancer tissue into the culture vessel coated with extracellular matrix gel and culturing them using the primary cell culture medium of step (1); A method comprising:

13. 1. A method for evaluating or screening a drug for treating lung cancer disease, comprising: (1) Culturing lung cancer epithelial cells by the culture method according to claim 12; (2) selecting the drug to be tested and diluting it into various drug concentration gradients; (3) adding the drug diluted in a gradient to the lung cancer epithelial cells obtained in step (1) and detecting cell viability; A method comprising:

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