RU2816529C1 - Culture medium for epithelial cells of esophageal squamous cell carcinoma, method of cultivation and use thereof - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention discloses a new culture medium containing MST1/2 kinase and ROCK kinase inhibitors and providing a shorter period of cell culture in comparison with known analogues. Invention can be used for cultivation or propagation in vitro of epithelial cells of primary squamous cell carcinoma of esophagus.

EFFECT: cell model obtained using the culture medium for primary cells according to the present invention can be used to assess the efficacy and screening of drugs suitable for treating oesophageal cancer.

14 cl, 14 dwg, 9 tbl, 8 ex

Description

Field of technology

The invention relates to the field of medical technology, in particular to a culture medium and a culture method for cultivating or propagating primary epithelial cells of squamous cell carcinoma of the esophagus in vitro, as well as a method and use of cultured cells in evaluating the effectiveness and screening of drugs.

State of the art

Esophageal cancer is one of the most common malignant neoplasms of the gastrointestinal tract in the world. According to the latest statistics from the National Cancer Center, among the ten most common malignant tumors, esophageal cancer ranks sixth in men and second in women. In many regions of the world, the local incidence rate has increased, with China being a region with a high incidence of esophageal cancer, with an annual incidence of 246,000 and an average annual death rate of about 150,000, accounting for 21.8% of the country's cancer mortality, ranking fourth among cancer types. causing the greatest number of deaths (data from the National Cancer Center, 2019). In recent years, although people have made some progress in the research on molecular typing and pathogenesis of esophageal cancer, due to the differences in molecular typing of esophageal cancer between China and foreign countries, therapeutic tools for esophageal squamous cell carcinoma (ESCC), particularly for patients with advanced stages are still limited, and personalized recommendations for personalized medicine are not available. Unlike adenocarcinoma, SCC does not have a relatively clear molecular target, and therefore, in practice, it is difficult to predict the effectiveness of clinical drugs based on molecular or genetic diagnosis alone.

Functional testing refers to a method of determining the sensitivity to anticancer drugs on cells from cancer patients in vitro. Key to the application of this method is the development of tumor cell models that have a short growth cycle and can reflect the biological characteristics of patients with SCC. In addition, the cell model must be easy to handle in order to quickly and effectively predict the effectiveness of clinical drugs, in order to provide timely and accurate treatment recommendations for patients with cancer. However, the generally low success rate of generating in vitro cell models from primary tumor cells of cancer patients, long growth cycles, and problems such as excessive proliferation of mesenchymal cells (such as fibroblasts, etc.) limit development in this field. There are currently two primary epithelial/stem cell culture technologies that are relatively mature developments in the field of functional testing using tumor cells. One is a technology that uses irradiated feeder cells and Y27632, a ROCK kinase inhibitor, to stimulate the growth of primary epithelial cells to study drug sensitivity in individual patients, that is, a condition-induced cell reprogramming technology (Liu et al., Am J Pathol, 180: 599-607, 2012). Another technique is 3D in vitro culture of adult stem cells to produce tissue- and organ-like organoids (Hans Clevers et al., Cell, 11; 172(1-2): 373-386, 2018).

However, both methods have certain limitations. Cell reprogramming is a technique in which the patient's primary autologous epithelial cells are co-cultured with mouse-derived feeder cells. The presence of these murine-derived cells may influence drug sensitivity test results on primary autologous patient cells during drug sensitivity testing on primary patient cells; on the other hand, in the case of knockout of mouse-derived feeder cells, the patient's primary autologous cells may be removed from the reprogramming environment, and the rate of cell proliferation and intracellular signaling may be significantly altered (Liu et al., Am J. Pathol, 183(6): 1862 -1870, 2013; Liu et al., Cell Death Dis., 9(7): 750, 2018), resulting in a significant impact on the response of the patient's primary autologous cells to the drug. Organoid technology is a technology in which primary autologous patient-derived epithelial cells are embedded in an extracellular matrix for 3D in vitro culture, which does not require feeder cells and thus avoids the problem of interference from mouse-derived feeder cells. However, the culture medium of organoid technology must be supplemented with various specific growth factors (such as Wnt proteins and R-spondin family proteins), which is expensive and not suitable for widespread clinical use. Additionally, the organoid must be encased in a gel-like extracellular matrix throughout the culture process, and the steps of cell inoculation, subculture, and drug sensitivity testing are labor-intensive and time-consuming compared to 2D culture operations. In addition, the size of the organoid formed using this technology is difficult to control, and some organoids may become too large and cause internal necrosis. Thus, organoid technology has poorer feasibility and applicability than 2D culture technology. It requires a technical professional to implement and is therefore not suitable for general and widespread clinical use for in vitro drug susceptibility testing (Nick Barker, Nat Cell Biol, 18(3): 246-54, 2016).

Due to the limitations of the above technologies, it is necessary to develop a technology for culturing primary epithelial cells of PCPC in the clinic, which can provide a short culture period, controlled cost and convenient operation without the influence (interference) of exogenous cells. When the technology is applied to create a primary RCC tumor cell model, the cultured RCC tumor cells can represent the biological characteristics of RCC patients. By assessing the sensitivity of anticancer drugs in vitro, in cell models derived from individual cancer patients, the response rate to anticancer drug therapy in the clinic can be improved, and problems caused by inappropriate drugs for patients and the consumption of medical resources can be reduced.

Brief description of the invention

Taking into account the shortcomings of the prior art, the invention is directed to the creation of a culture medium for culturing primary epithelial cells of SCC and a method for culturing primary epithelial cells of SCC using the specified culture medium. The culture medium for primary SCLC epithelial cells and the culture method of the present invention can provide a short in vitro culture period, controlled cost, convenient operation and no interference from exogenous cells. By applying this technology to create a primary PCR tumor cell model, it is possible to obtain primary PCR tumor cells with the biological characteristics of PCR patients, which can be used in new drug screening and in vitro drug sensitivity testing.

One aspect of the invention is to provide a culture medium for culturing primary epithelial cells of SCRC containing an MST1/2 kinase inhibitor and a ROCK kinase inhibitor, wherein the MST1/2 kinase inhibitor contains a compound of Formula (I) or a pharmaceutically acceptable salt or solvate thereof, and the ROCK kinase inhibitor is at least one selected from the group consisting of Y27632, Fasudilai H-1152.

Where

R ₁ is selected from C1-C6 alkyl, C3-C6 cycloalkyl, C4-C8 cycloalkylalkyl, C2-C6 spirocycloalkyl and aryl (e.g. phenyl and naphthyl, etc.), optionally independently containing 1-2 R ₆ as substituents, aryl-C1-C6 alkyl (for example, phenylmethyl, etc.), optionally independently containing 1-2 R ₆ as substituents, and heteroaryl (for example, thienyl, etc.), optionally independently containing 1- 2 R ₆ ;

R ₂ and R ₃ are each independently selected from C1-C6 alkyl, preferably C1-C3 alkyl, more preferably methyl;

each of R ₄ and R ₅ independently selected from hydrogen, C1-C6 alkyl, C3-C6 cycloalkyl, C4-C8 cycloalkylalkyl, hydroxyl, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkylamino C1-C6 alkyl, C1 -C6 alkoxy C1-C6 alkyl and C3-C6 heterocyclyl-C1-C6 alkyl (heterocyclyl is selected, for example, from piperidyl, tetrahydropyranyl, etc.);

R ₆ is selected from halogen (preferably fluorine and chlorine, more preferably fluorine), C1-C6 alkyl (preferably methyl), C1-C6 alkoxy (preferably methoxy) and C1-C6 haloalkyl (preferably trifluoromethyl).

In a preferred embodiment of the invention, the MST1/2 kinase inhibitor contains a compound of Formula (Ia) or a pharmaceutically acceptable salt or solvate thereof,

wherein

R ₁ is selected from C1-C6 alkyl, phenyl, optionally independently containing 1-2 R ₆ as substituents, thienyl, optionally independently containing 1-2 R ₆ as substituents, and phenylmethyl, optionally independently containing 1-2 R as substituents ₆ ; more preferably, R ₁ is phenyl, optionally independently substituted with 1-2 R ₆ ;

R ₅ is selected from hydrogen, C1-C6 alkyl and C3-C6 cycloalkyl; more preferably, R ₅ is hydrogen;

R ₆ is independently selected from halogen, C1-C6 alkyl and C1-C6 haloalkyl; more preferably, R ₆ is fluoro, methyl or trifluoromethyl.

Preferably, the MST1/2 kinase inhibitor is at least one compound selected from the following compounds or a pharmaceutically acceptable salt or solvate thereof.

Most preferably, the MST1/2 kinase inhibitor of the invention is Compound 1, and the ROCK kinase inhibitor of the invention is preferably Y27632.

In an embodiment of the invention, the amount of MST1/2 kinase inhibitor in the culture medium is typically 0.3 μM-10 μM, preferably 0.75 μM-6 μM, more preferably 2 μM-6 μM. In addition, the amount of ROCK kinase inhibitor in the culture medium is usually 0.3-20 μM, preferably 2.5-15 μM, more preferably 7.5-12.5 μM.

Preferably, the primary cell culture medium of the invention further contains one or more of the following factors: epidermal growth factor (EGF); insulin-transferrin-selenium complex; additive B27 or additive N2; hepatocyte growth factor (HGF); a type I TGFp receptor inhibitor selected from at least one of A83-01, SB431542, Repsox, SB505124, SB525334, SD208, LY36494 and SJN2511; and a P38/MAPK inhibitor selected from at least one of SB202190, SB203580, VX-702, VX-745, PD169316, RO4402247, and BIRB796.

In a preferred embodiment, the amount of EGF is from 12.5 ng/ml to 100 ng/ml, more preferably from 50 ng/ml to 100 ng/ml; the corresponding amounts of insulin/transferrin/sodium selenite in the insulin-transferrin-selenium complex are 2.5-20 μg/ml, 1.25-10 μg/ml, 1.25-10 ng/ml, respectively, and more preferably 5 -20 µg/ml, 2.5-10 µg/ml, 2.5-10 ng/ml, respectively; the volume concentration of additive B27 or additive N2 in the culture medium is 1:25-1:200, more preferably 1:25-1:50; the amount of HGF is 2.5 ng/ml-20 ng/ml, more preferably 10 ng/ml-20 ng/ml; the type I TGFβ receptor inhibitor is preferably A83-01, and the amount of the type I TGFβ receptor inhibitor is 125nM-500nM, preferably 250nM-500nM; the P38/MAPK inhibitor is preferably SB202190, and the amount of the P38/MAPK inhibitor is 125 nM to 500 nM, preferably 250 nM to 500 nM.

Compared to Condition-Induced Cell Reprogramming Medium and CCPC Epithelial Cell Organoid Medium, this media formulation contains a combination of an MST1/2 kinase inhibitor and a ROCK kinase inhibitor (Compound 1 and Y27632 are particularly preferred) but does not contain unidentified components such as as: whey, bovine pituitary gland extract or similar; niche factors that are necessary for organoid culture, such as Wnt agonists, R-spondin family proteins, BMP inhibitors or the like; and also does not contain nicotinamide and N-acetylcysteine, which significantly reduces the cost of the medium, simplifies the workflow for preparing the medium and obtaining a culture of primary epithelial cells of SCC in vitro due to controlled costs and ease of handling.

In the present invention, the primary PCR epithelial cells can be selected from PCR tumor cells, normal PCR epithelial cells, and PCR epithelial stem cells.

One aspect of the invention is a method for culturing primary epithelial cells of SCC, comprising the following steps:

(1) Preparation of the culture medium for primary cells according to the invention according to the composition described above.

(2) Coating the culture vessel with a diluted gel-like extracellular matrix.

In particular, a gelled extracellular matrix with low growth factor content can be used as the gelled extracellular matrix, for example, commercial Matrigel (purchased from Corning) or BME (purchased from Trevigen) can be used. More specifically, the gelled extracellular matrix is diluted with serum-free medium, which may be DMEM/F12 medium (purchased from Corning). The dilution rate of the gelled extracellular matrix is 1:50-1:400, preferably 1:100-1:200. The coating method involves adding a diluted gel-like extracellular matrix to a culture vessel to completely cover the bottom of the culture vessel, and maintaining for 30 minutes or more, preferably maintaining and coating at 37°C for 30-60 minutes. Once coating is complete, excess diluted extracellular matrix gel is discarded and the culture vessel is ready for further use.

(3) Isolation of primary SCRC epithelial cells from SCRC tissue. Primary SCRC epithelial cells can be obtained, for example, from SCRC tissue samples and peritumoral tissue samples. For example, PCR tissue samples are obtained from the tumor tissue of an informed and consenting PCR patient by surgical resection, and peritumoral tissue samples are collected from PCR tissue at least 5 cm from the esophageal tissue. The above tissue samples are collected within half an hour after surgical excision or biopsy. More specifically, a tissue sample is excised under sterile conditions from non-necrotic areas greater than 0.5 mm ³ in volume, and then the tissue sample is placed in 10-15 ml of pre-chilled DMEM/F12 medium, placed in a plastic sterile centrifuge tube with a cap, and transported to the laboratory on ice. In this case, the DMEM/F12 medium contains 1-2 vol.% penicillin, streptomycin and/or 0.2-0.4 vol.% Primocin (hereinafter referred to in this document as tissue transport fluid). In the case of using streptomycin/penicillin, the concentration range of streptomycin is 25-400 µg/ml, preferably 50-200 µg/ml, more preferably 200 µg/ml, the concentration range of penicillin is 25-400 U/ml, preferably 50-200 U/ml , more preferably 200 U/ml; and in the case of Primocin, the concentration range is 25-400 µg/ml, preferably 50-200 µg/ml, more preferably 100 µg/ml.

In a biological safety cabinet, the tissue sample is transferred to a cell culture dish, which is then rinsed with a transport fluid to wash away blood cells from the surface of the tissue sample. The washed tissue sample is transferred to another new culture dish with the addition of 1-3 ml of transport liquid and, using a sterile scalpel blade and tweezers, the tissue sample is divided into tissue fragments with a volume of less than 3 mm ³ .

The tissue sample fragments are transferred to a centrifuge tube, which is centrifuged at 1500 rpm for 3-5 minutes in a benchtop centrifuge (Sigma, 3-18K); after removing the supernatant, add tissue transport fluid and a solution for enzymatic disaggregation of tissues in a 1:1 ratio (the dosage is about 5 ml of a solution for enzymatic treatment of tissues per 10 mg of tissue; the method of preparing a solution for enzymatic disaggregation of tissues includes: dissolving 1-2 mg/ml collagenase II, 1-2 mg/ml collagenase IV, 50-100 U/ml deoxyribonucleic acid I, 0.5-1 mg/ml hyaluronidase, 0.1-0.5 mg/ml calcium chloride, 5-10 mg/ ml bovine serum albumin in HBSS and RPMI-1640 in a volume ratio of 1:1); then the sample is numbered and sealed with a sealing film, and then subjected to enzymatic disaggregation in a constant temperature shaker (Zhichu Instrument ZQLY-180N) at 37°C, with rotation of 200-300 revolutions; completeness of cleavage is determined by observation every 1 hour; if no obvious tissue fragment is detected, the enzymatic disaggregation process can be stopped; otherwise, the sample continues to undergo enzymatic disaggregation until tissue digestion is sufficient, with enzymatic disaggregation times varying from 4 to 8 hours. After enzymatic disaggregation, the undigested tissue fragments are filtered through a cell mesh filter (the mesh size of the cell mesh filter is, for example, 70 μm); tissue fragments on the filter mesh are washed with tissue transport fluid; the remaining cells are washed into a centrifuge tube and centrifuged in a benchtop centrifuge at 1500 rpm for 3-5 minutes. After removing the supernatant, the remaining cell clumps are observed to determine whether blood cells remain; if blood cells are present, add 3 ml of blood cell lysate (purchased from Sigma), which is then mixed well, lysed at 4°C for 10-20 minutes, shaking and mixing thoroughly every 5 minutes; after lysis, the resulting product is removed and centrifuged at 1500 rpm for 3-5 minutes. After removing the supernatant, the basic culture medium according to the invention is added for resuspension. The total number of cells is obtained by counting with a flow cell imaging counter (JIMBIO FIL, Jiangsu Jimbio Technology Co., Ltd.).

(4) Inoculating the primary culture SCPC epithelial cells isolated in step (3) into a coated culture vessel and culturing using the primary culture cell culture medium obtained in step (1).

Namely, tumor cells of the primary culture of SCC are inoculated at a density of 2×10 ⁴ to 8×10 ⁴ cells/cm ² (for example, 4×10 ⁴ cells/cm ² ) into one well of a 6-well plate; add 2-3 ml of basic medium for culturing epithelial cells, then culture in a cell incubator for 8-16 days, for example, under conditions of 37°C, 5% CO ₂ ; every 4 days during cultivation, the medium for the primary culture cells is changed to fresh; Enzymatic disaggregation and passaging are performed when primary SCPC epithelial cells have grown to a cell density that is 80% to 90% of the bottom area of a 6-well plate.

This inoculation step does not require the use of feeder cells, and, compared to condition-induced cell reprogramming technology, the steps of culturing and irradiating feeder cells are omitted. Compared to organoid technology, this step does not require uniformly mixing primary culture cells with a gel matrix on ice to form gel droplets and waiting for the gel droplets to solidify before adding media. A pre-coated culture vessel can be used directly to inoculate primary culture cells. In addition, only a small amount of diluted extracellular matrix gel is required to coat the culture vessels, resulting in savings on expensive extracellular matrix gel and simplified operations compared to organoid technology.

Optionally, after culturing the inoculated primary SCPC epithelial cells for 8-16 days, when the cell clones formed in the culture container converge and cover 80% of the bottom area, the supernatant is removed and 1-2 ml of 0.05% trypsin (purchased from Thermo Fisher) is added ) to enzymatically disaggregate the cells, which are then incubated at room temperature for 5-20 minutes. Enzyme-treated cells are resuspended in 2-4 ml of culture fluid containing, for example, 5% (v/v) fetal bovine serum, 100 U/ml penicillin and 100 μg/ml streptomycin, and centrifuged at 1500 rpm in for 3-5 minutes. The disaggregated single cells are resuspended using the primary cell culture medium of the invention, and the resulting cell suspension is placed in a T25 culture flask coated with extracellular matrix gel for continuous culture. The operation of coating the T25 culture flask is the same as in step (2).

Expanded SCPC epithelial cells grow in a 2D format, without the formation of heterogeneously sized organoids and internal necrosis in expanded organoids that can occur when expanded using organoid technology.

In another aspect, PCR epithelial cells, especially PCR tumor cells grown by the primary PCR epithelial cell culture method of the invention, can be used for drug efficacy evaluation and drug screening, which includes the following steps:

(1) obtaining primary RCC epithelial cells, more preferably, obtaining tumor tissue samples or tumor tissue biopsy samples taken from patients with RCC; isolating primary PCR epithelial cells; and culturing and expanding primary PCR epithelial cells (in particular, primary PCR tumor cells) in accordance with the method for culturing primary SCRC epithelial cells described above until the number of cells reaches 10 ⁵ , preferably 10 ⁶ .

(2) Selection of drug to be tested.

(3) Based on the maximum plasma drug concentration _Cmax as the reference, use 2-5 times the _Cmax value as the initial concentration, and dilute the drug using different concentration gradients, such as 5-10 drug concentration gradients , preferably 6-8.

(4) Cleavage (disaggregation) of the PCR epithelial cells cultured in step (1) into a suspension of individual cells; counting the number of cells using an in-line imaging counter; diluting the single cell suspension with a primary cell culture medium according to the invention, adding the diluted cell suspension evenly into a multiwell plate at a density of 2000-4000 cells per well, for example, 50 μl of the diluted cell suspension per well; and cell attachment overnight.

This step avoids the problem of the cell reprogramming method that the presence of feeder cells may interfere with the counting of primary culture cells and subsequent analysis of primary culture cell viability; and also eliminates the need for the tedious step of mixing, embedding and then seeding the cell suspension into a gel-like matrix on ice, as in organoid technology, which greatly simplifies the process and increases the performance and practicality of the technology. Because the inoculated cells are single cell suspensions rather than three-dimensional structures such as organoids, this method can produce more uniform cell counts across plates and less variation in cell counts between wells compared to organoid technology, making this method more suitable for subsequent high-throughput drug screening operations.

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

(6) A few hours after drug addition, eg, 72 hours, determine the survival rate of SCPC epithelial cells using a Cell-Titer Glo Luminescence Cell Viability Kit (purchased from Promega) to screen for drug activity.

Specifically, for example, 10 μl of Cell Titer-Glo reagent (purchased from Promega) is added to each well, and after uniform shaking, the chemiluminescence intensity of each well is measured using a fluorescence microplate reader. Taking the drug concentration as the abscissa and the fluorescence intensity as the ordinate, GraphPad Prism software is used to construct a drug dose-response curve based on the measured values, and the inhibitory activity of the drug on the proliferation of the test cells is calculated.

When using primary PCR tumor cells according to the invention for drug screening and in vitro drug sensitivity testing, the phenomenon that feeder cells in the cell reprogramming technology interfere with the test results does not occur because there is no cell co-culture in this system. Due to cell growth in a 2D plane, drug interaction is also faster than drug testing time in organoid technology (average exposure time in organoid technology is 6 days).

The beneficial effects of the invention also include:

(1) the success rate of culturing primary SCPC epithelial cells can be improved, with a success rate of more than 80%;

(2) epithelial cells of PCPC, primary cultured in vitro, can be obtained to reproduce the pathological phenotype and heterogeneity of the patient-host cells of the primary culture;

(3) primary SCRC epithelial cells are pure SCRC epithelial cells that are not interfered with by fibroblasts;

(4) the composition of the medium does not contain serum, which means it is not affected by the quality and quantity of serum from different batches;

(5) PCR epithelial cells can be expanded with high efficiency, up to a value of 10 ⁶ PCR epithelial cells, in this case, the cells were successfully expanded within about two weeks after the start of cell counting at 10 ⁴ , and the grown PCR epithelial cells have the ability to continuously passivation;

(6) there is no need to work on ice and dissociate the gel matrix at the subculture step, and cell digestion and subculture can be completed within 10-15 minutes;

(7) The cost of culture is controllable because the culture medium for primary PCR does not require expensive Wnt agonists, R-spondin family proteins, BMP inhibitors and similar factors, and thus it is a simplification and improvement of the existing culture medium for organoids for primary epithelial cells PKRP; and when inoculating cells, it is not necessary to use a higher concentration of extracellular matrix to mix with primary cells to form gel droplets, but instead only requires a small amount of dilute gelled extracellular matrix, which saves the amount of expensive extracellular matrix;

(8) Operations are convenient: Compared with condition-induced cell reprogramming technology, this technology does not require the cultivation or irradiation of feeder cells, which avoids the problem that the quality and quantity of feeder cells from different batches may affect the efficiency of the primary cell culture ; and the objects seeded and tested in drug screening are only primary epithelial cells of SCRC, without interfering feeder cells in the co-culture system, which are present in the technology of condition-induced cell reprogramming; Compared with the organoid culture technology, in the gel-like extracellular matrix coating method adopted in the invention, the culture vessel can be prepared in advance, and there is no need to enclose the cells in the gel-like matrix as in the organoid technology; also, the operation steps are simple and easy;

(9) This technology allows the cultivation and provision of PCR epithelial cells in large numbers and with high homogeneity, which is suitable for high-throughput screening of new drug candidates and high-throughput in vitro functional drug susceptibility testing for patients.

Using the cell culture medium of this embodiment of the present invention, it is possible to culture SSCC epithelial cells obtained from humans or other mammals, including SSCC tumor cells, normal esophageal epithelial cells, SSCC epithelial stem cells, or tissues containing at least any of these cells. At the same time, the culture medium according to the invention can also be used to develop a kit for propagation and culturing of primary cultured SCPC cells in vitro.

In addition, the cells obtained by the culture method according to this embodiment of the present invention can be used in regenerative medicine, basic medical research on SCC epithelial cells, drug response screening and development of new drugs for SCC diseases, and the like.

Description of drawings

Fig. 1A-1E show the influence of various factors of the culture medium on the proliferation of cells in the primary culture of PCR; in Fig. 1F shows the effect of culture medium containing a combination of compound 1 and Y27632 at various concentrations on the proliferation of primary cultured SCRC cells.

In FIG. Figure 2 shows the influence of increasing amounts of factors in the culture medium on the proliferation of cells in the primary culture of SCRC.

Fig. 3A-3H show the effect of the concentration of each factor on the proliferation of cells in the primary culture of SCC.

Fig. 4A and 4B are inverted microscope photographs of PPCC tumor cells that were grown from cells isolated from one clinical specimen of PPCC tissue (#0F0062) using the FEM culture medium of the invention until day 4 and day 12, respectively.

Fig. 5A and 5B are microscopic photographs of cells that were grown from primary RCC tumor cells derived from one RCC tissue (#0F0065) until day 11, under Matrigel gelled extracellular matrix coating conditions and without Matrigel coating, respectively.

FIGS. 6A-6E are inverted microscope photographs of cells that were grown from cells isolated from one surgically removed SCC specimen (#0F0060) for 14 days in five different culture media.

Fig. 7 is a comparative chart of the effects of cell proliferation obtained by culturing cells isolated from six surgically removed SCLC specimens (#0F0056, 0F0060, 0F0061, 0F0062, 0F0071, 0F0075) for 16 days in five different culture media.

Fig. 8 is a comparative diagram of cell growth curves obtained by culturing cells isolated from one clinical sample of SCC tissue (#0F0075) under five different culture media.

Fig. 9A-9D are microscopic photographs of cells grown from cells isolated from one clinical SCLC tissue sample (#0E0075) up to the 1st and 9th passages, under FEM and EPI culture conditions, respectively.

Fig. 10A and 10B are a comparison of the immunohistochemistry results of one surgically removed SCC specimen (#0F0053) and SCC tumor cells obtained by culturing cells isolated from the sample (#0F0053) with the FEM culture medium of the invention, respectively.

Fig. 11A-11D are a comparison of the results of copy number analysis of SCC tumor cells in one surgically removed SCC specimen (#0F0025) and after different subcultures obtained by culturing cells isolated from the sample (#0F0025) with the FEM culture medium of the invention, respectively.

Fig. 12 is a comparison chart of cell growth curves obtained by culturing cells isolated from one clinical SCC tissue sample (No. 0F0061) under the conditions of the FEM culture medium according to the invention and the culture media obtained by subtracting various components, respectively.

Fig. 13A-13H are microscopic photographs of cells obtained by culturing cells isolated from one clinical sample of SCRC tissue (No. 0F0061), before the third subculture, under the conditions of the FEM culture medium according to the invention and the culture media obtained by subtracting various components, respectively.

In FIG. 14A and 14B show dose-response curves of tumor cells from a primary culture of RCC to various chemotherapeutic drugs and targeted drugs, wherein tumor cells of a primary culture of RCC were obtained by growing from surgically removed tumor tissue samples from two different patients with RCC (#0F0060 and No. 0F0062) with the FEM culture medium according to the invention, respectively.

Detailed Description of the Invention

In the description of characteristics, epithelial cells include differentiated epithelial cells and epithelial stem cells derived from epithelial tissues. The term "Epithelial stem cells" refers to cells with long-term self-renewal capacity that can differentiate into epithelial cells, and to stem cells derived from epithelial tissues. Examples of epithelial tissues include: cornea, oral mucosa, skin, conjunctiva, bladder, renal tubules, kidneys, digestive organs (esophagus, stomach, duodenum, small intestine (including jejunum and ileum), colon (including colon )), liver, pancreas, mammary gland, salivary gland, lacrimal gland, prostate gland, hair root, trachea, lung, etc. Among others, the cell culture medium (cell culture medium) according to this embodiment of the present invention is preferably is a medium for culturing epithelial cells obtained from the esophagus.

Moreover, as used herein, the term “epithelial tumor cells” refers to cells derived from tumorigenesis of cells derived from the aforementioned epithelial tissues.

In the description of characteristics, the term "organoid" refers to a three-dimensional organ-like cellular tissue formed by the spontaneous organization and aggregation of cells in a controlled space at high density.

[Examples of preparation of MST1/2 kinase inhibitors]

In the description of characteristics, an MST1/2 kinase inhibitor refers to any inhibitor that directly or indirectly negatively regulates MST1/2 signaling. In general, MST1/2 kinase inhibitors reduce the activity of MST1/2 kinase, for example by binding to it. Since MST1 and MST2 have a similar structure, MST1/2 kinase inhibitors could be, for example, compounds that bind to MST1 or MST2 and reduce their activity.

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)benzensulfamide 1

Methyl 2-amino-2-(2,6-difluorophenyl)acetate (A2): 2-amino-2-(2,6-difluorophenyl)acetic acid (2.0 g) and then methanol (30 ml) were added to the round bottom laboratory flask and then add thionyl chloride (1.2 ml) dropwise in an ice bath. The reaction of the reaction system was carried out overnight at 85°C. After completion of the reaction, the system was evaporated under reduced pressure to dry the solvent, and the resulting white solid was directly used in the next step.

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

2-chloro-7-(2,6-difluorophenyl)-7,8-dihydropteridin-6(5H)-one (A4): methyl 2-((2-chloro-5-nitropimidin-4- yl)amino)-2-(2,6-difluorophenyl)acetate (2.5 g), then acetic acid (50 ml) and iron powder (3.9 g). The reaction system was stirred at 60°C for two hours. After completion of the reaction, the reaction system was evaporated under reduced pressure to dry the solvent, and 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 brine and dried with anhydrous sodium sulfate. The organic phase was filtered and evaporated to dryness under reduced pressure to obtain the crude product. The crude product was washed with diethyl ether to obtain compound A4. Liquid chromatography-mass spectrometry, LC/MS: M+H 297.0.

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 laboratory flask and cooled to -35°C, followed by the addition of iodomethane (0.9 ml), and then sodium hydride (615 mg), and the reaction system was stirred for two 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 brine, respectively, and dried with anhydrous sodium sulfate. The organic phase was filtered and evaporated to dryness under reduced pressure to obtain the crude product. The crude product was washed with diethyl ether to obtain compound A5. Liquid chromatography-mass spectrometry, LC/MS: M+H 325.0.

4-((7-(2,6-difluorophenyl)-5,8-dimethyl-6-oxo-5,6,7,8-tetrahydropteridin-2-yl)amino)benzensulfamide (1): 2-chloro-7 -(2,6-difluorophenyl)-5,8-dimethyl-7,8-dihydropteridin-6(5H)-one (100 mg), sulfonamide (53 mg), p-toluenesulfonic acid (53 mg) and secondary butanol (5 ml) was added to a round-bottomed laboratory flask. The reaction system was stirred at 120°C overnight. After completion of the reaction, the reaction mixture was filtered and washed with methanol and diethyl ether to obtain compound 1. Liquid chromatography-mass spectrometry, LC/MS: M+H 461.1.

2. Preparation of other MST1/2 inhibitor compounds according to the invention.

Other MST1/2 inhibitor compounds of the invention were synthesized in a manner similar to the synthesis of Compound 1, and their structures and mass spectrum data are shown in the following table.

[Example 1]

Isolation of primary human SCC epithelial cells

SCC tissue samples were obtained from tumor tissues of three informed and consenting patients with SCC that were surgically removed, namely, specimens #0F0060, 0F0061, and 0F0062. One of the samples (No. 0F0060) will be described below.

The above tissue samples were collected within half an hour of surgical excision or biopsy. Specifically, under sterile conditions, tissue samples were excised from non-necrotic areas greater than 0.5 cm ³ in volume and placed in 4 ml of pre-cooled tissue transport fluid (specific composition shown in Table 1). The transport liquid was placed into a plastic sterile cryopreservation tube with a cap (purchased from Guangzhou Jet Bio-Filtration Co., Ltd.) and transported to the laboratory using a cold chain system (0-10°C).

In a biosafety laminar flow hood, the tissue sample (#0F0060) was transferred to a 100 mm diameter cell culture dish (purchased from NEST). The tissue sample was washed with tissue transport fluid. Any remaining blood on the surface of the tissue sample was washed off. Excess tissue, such as fat on the surface of the tissue sample, was removed. The washed tissue sample was transferred to another new culture dish with a diameter of 100 mm; 2 ml of transport fluid was added and a sterile scalpel blade and tweezers were used to separate the tissue sample into tissue fragments with a volume of less than 3 mm ³ .

Tissue sample fragments were transferred to a 15 ml centrifuge tube and centrifuged at 1500 rpm for 4 minutes in a benchtop centrifuge (Sigma, 3-18K); after removing the supernatant, tissue transport fluid and enzymatic tissue disaggregation solution were added in a 1:1 ratio (the dosage is about 5 ml of enzymatic tissue disaggregation solution per 10 mg of tissue; the exact composition is shown in Table 2); the sample was then numbered and sealed with a sealing film, and then kept in a constant temperature shaker (Zhichu Instrument ZQLY-180N) at 37°C, 300 revolutions; the completeness of enzymatic disaggregation was determined by monitoring every 1 hour.

After enzymatic disaggregation, undigested tissue fragments were filtered through a 70-μm cell mesh filter; tissue fragments on the filter mesh were washed with tissue transport fluid; the remaining cells were washed into a centrifuge tube and centrifuged at 1500 rpm for 4 minutes.

After removing the supernatant, the remaining cell clumps were observed to determine whether blood cells remained; if blood cells were present, 3 ml of blood cell lysate (purchased from Sigma) was added, which was then mixed well, lysed at 4°C for 15 minutes, with shaking and thorough mixing every 5 minutes; After lysis, the resulting product was recovered and centrifuged at 1500 rpm for 4 minutes. The supernatant was discarded to obtain disaggregated and isolated cells from the primary PCR culture, to which basal medium (BM) was added for resuspension. Basic medium was prepared by adding 0.2 vol% Primocin (purchased from Invivogen, 50 mg/ml concentration) to commercial DMEM/F-12 medium to obtain a final concentration of 100 μg/ml. The total number of cells was 2,280,000, which was obtained by counting with a flow cell imaging counter (JIMBIO FIL, Jiangsu Jimbio Technology Co., Ltd.).

Two other PCR tumor tissue samples were isolated according to the same method described above, and the total cell numbers were 1,470,000 (0F0061) and 2,680,000 (0F0062), respectively.

[Example 2]

Optimization of culture medium for primary epithelial cells of SCRC (1) Influence of various factors

Gel-like extracellular matrix (Matrigel®) (manufactured by BD Biosciences) was diluted 1:100 in serum-free DMEM/F12 to prepare an extracellular matrix solution, which was added to a 48-well culture plate at a rate of 500 μl/well until completely covered. the bottom of the wells of the culture plate. The resulting mixture was left for 1 hour in an incubator at 37°C. After 1 hour, the extracellular matrix solution was removed to obtain a Matrigel matrix-coated plate.

Preparation of basal medium (abbreviated BM): BM was prepared by adding 0.2 vol% Primocin (purchased from Invivogen, 50 mg/mL concentration) to commercial DMEM/F-12 medium to obtain a final concentration of 100 μg/mL.

Various types and concentrations of additional factors (Table 3) were then added to the basal medium (BM) to prepare culture media for SCLC epithelial cells containing various additional components.

Culture media with various components were added in a volume of 500 μl/well to 48-well plates coated with a gel-like extracellular matrix (Matrigel). SCC tumor cells (No. 0F0064), isolated from SCC tissue according to the same method described in Example 1, were inoculated into a 48-well culture plate coated with Matrigel matrix at a cell density of 2×10 ⁴ cells/cm ² . After surface disinfection, the plate was placed in a 37°C, 5% CO ₂ incubator (purchased from Thermo Fisher), such that an equal number of freshly isolated SCC tumor cells (#0F0064) were cultured in different media compositions. Culture media were renewed every 4 days after the start of cultivation. After 12 days of cultivation, cell counts were performed. The basal medium (BM) without any additives was used as a control for the experiment. The results are presented in Fig. 1A-1E. The ordinate in each figure represents the ratio of the number of cells obtained after cultivation in various media to the number of cells obtained after cultivation in the main VM medium. As shown in the figures, adding different concentrations of different factors as shown in Table 3 to the HMW medium had different effects on cell proliferation. Among others, Supplement B27, Supplement N2, insulin-transferrin-selenium complex, epidermal growth factor, hepatocyte growth factor, insulin-like growth factor 1, Compound 1 and Y27632 have a stimulating effect on cell proliferation in certain concentration ranges.

(2) Effects of the combination of Y27632 and Compound 1.

Gel-like extracellular matrix (Matrigel®) (purchased from BD Biosciences) was diluted 1:100 in serum-free DMEM/F12 to prepare an extracellular matrix solution, which was added to a 48-well culture plate at a rate of 500 μl/well until the bottom of the culture wells was completely covered. tablet. The resulting mixture was left for 1 hour in an incubator at 37°C. After 1 hour, the extracellular matrix solution was removed to obtain a Matrigel matrix-coated plate.

Various concentrations of Y27632 and Compound 1 (Table 4) were added to the BM basal medium, thereby obtaining culture media for SCC epithelial cells containing various additional components.

Culture media with various components were added in a volume of 500 μl/well to 48-well plates coated with a gel-like extracellular matrix (Matrigel). SCC tumor cells (No. 0F0063), isolated from SCC tissue according to the same method as described in Example 1, were inoculated into a 48-well culture plate coated with Matrigel matrix at a cell density of 2×10 ⁴ cells/cm ² . After surface disinfection, the plate was placed in a 37°C, 5% CO ₂ incubator (purchased from Thermo Fisher), so that an equal number of freshly isolated SCC tumor cells (#0F0063) were cultured in different media compositions. Culture media were renewed every 4 days after the start of cultivation. After 12 days of cultivation, cell counts were performed. The main VM environment was used as a control for the experiment. The results are presented in Fig. 1F. As shown in the figure, combinations of different concentrations of Y27632 and Compound 1 have a certain synergistic effect on cell proliferation, with the combination of 10 μM Y27632 and 3 μM Compound 1 having the most preferential effect.

(3) The influence of increasing amounts of factors in culture media on the proliferation of primary SCRC cells obtained by the method according to the invention

Gel-like extracellular matrix (Matrigel®) (purchased from BD Biosciences) was diluted 1:100 in serum-free DMEM/F12 to prepare an extracellular matrix solution, which was added to a 48-well culture plate at a rate of 500 μl/well until the bottom of the culture wells was completely covered. tablet. The resulting mixture was left for 1 hour in an incubator at 37°C. After 1 hour, the extracellular matrix solution was removed to obtain a Matrigel matrix-coated plate.

Various small molecules, additives, and growth factors (Table 5) were sequentially added to the BM basal medium, respectively, to obtain culture media for SCLC epithelial cells containing various additional components.

Culture media with various components were added in a volume of 500 μl/well to 48-well plates coated with a gel-like extracellular matrix (Matrigel), while VM medium was used as a control for the experiment. SCC tumor cells (No. 0F0065), isolated from SCC tissue according to the same method described in Example 1, were inoculated into a 48-well culture plate coated with Matrigel matrix at a cell density of 2×10 ⁴ cells/cm ² . After surface disinfection, the plate was placed in a 37°C, 5% CO ₂ incubator (purchased from Thermo Fisher), so that an equal number of freshly isolated SCC tumor cells (No. 0F0065) were cultured in different media compositions. After 10 days of cultivation, cell counts were performed. The results are presented in Fig. 2. As shown in the figure, No. 8 medium has been determined to be the most preferred culture medium in this patent for culturing and propagating primary SCC cells (hereinafter abbreviated as FEM). Based on this, further addition of certain factors or small molecule inhibitors did not have a significant effect on stimulating cell proliferation.

(4) Effects of different concentrations of additional factors on the proliferation of primary cultured PCR cells obtained in the invention

Gel-like extracellular matrix (Matrigel®) (manufactured by BD Biosciences) was diluted 1:100 in serum-free DMEM/F12 to create an extracellular matrix solution, which was added to a 48-well culture plate at 200 μl/well until completely covered. the bottom of the wells of the culture plate. The resulting mixture was left for 1 hour in an incubator at 37°C. After 1 hour, the extracellular matrix solution was removed to obtain a Matrigel matrix-coated plate.

Preparation of a culture medium for primary epithelial cells of PCR according to the invention (abbreviated as FEM): to the basic medium (BM) was added: epidermal growth factor EGF to a final concentration of 50 ng/ml, hepatocyte growth factor HGF to a final concentration of 10 ng/ml, stock solution with insulin-transferrin-selenium (ITS) complex to a final dilution of 1:50 (insulin to a final concentration of 10 μg/ml, transferrin to a final concentration of 5 μg/ml and sodium selenite to a final concentration of 5 ng/ml in FEM medium), additive B27 to a final dilution in a ratio of 1:50, compound 1 to a final concentration of 3 μM, Y27632 to a final concentration of 10 μM, TGFβ1 inhibitor A83-01 to a final concentration of 500 nM and P38/MAPK inhibitor SB202190 to a final concentration of 500 nM for the preparation of culture media for primary epithelial cells of PCR.

Tumor tissue-derived RCC epithelial cells were isolated and obtained from the tumor tissue of an RCC patient (sample No. 0F0065) using the same method as in Example 1. Then, tumor tissue-derived RCC epithelial cells were counted using a flow cell imaging counter (JIMBIO FIL, Jiangsu Jimbio Technology Co., Ltd.) to obtain the total number of cells. Cells were then inoculated into a Matrigel® matrix-coated (purchased from BD Biosciences) 12-well plate at a density of 4 x 10 ⁴ cells/cm ² . 2 ml of prepared FEM culture medium for primary epithelial cells of SCRC was added to a 12-well plate, which was then placed in culture conditions at 37°C, 5% CO ₂ in an incubator (purchased from Thermo Fisher). When the cells in the culture plate had grown to cover about 80% of the bottom surface area, the medium supernatant was removed and 500 μl of 0.05% trypsin (purchased from Gibco) was added to the 12-well plate to disaggregate the cells; cells were incubated at 37°C for 10 minutes until complete cell disaggregation, which was observed under a microscope (EVOS M500, Invitrogen); then enzymatic disaggregation was stopped using 1 ml of DMEM/F12 culture solution containing 5% (v/v) fetal bovine serum, 100 U/ml penicillin and 100 μg/ml penicillin; the resulting mixture was collected in a 15 ml centrifuge tube and centrifuged at 1500 rpm for 4 minutes, and then the supernatant was removed. The centrifuged cell pellets were resuspended in basal BM medium and counted using a flow cell imaging counter (JIMBIO FIL, Jiangsu Jimbio Technology Co., Ltd.) to obtain the total cell number. The resulting cells were used in the following culture experiments.

Next, the following 8 culture media with different compositions were prepared for the experiments:

Composition 1: FEM medium composition without B27 additive;

Composition 2: composition of FEM medium without the insulin-transferrin-selenium complex;

Composition 3: FEM medium composition without epidermal growth factor;

Composition 4: composition of FEM medium without hepatocyte growth factor;

Composition 5: FEM medium composition without Y27632;

Composition 6: FEM medium composition without Compound 1;

Composition 7: FEM medium composition without A83-01;

Composition 8: FEM medium composition without SB202190.

The suspension of disaggregated cells was diluted with the above compositions 1-8, respectively, and seeded into a 48-well plate in an amount of 10,000 cells and a volume of 250 μl per well.

When using Composition 1 medium, the prepared B27 supplement was added to a 48-well plate with inoculated primary culture cells at 250 μl per well so that the final concentrations of B27 supplement were 1:200, 1:100, 1:50 and 1:25, respectively; Composition 1 medium was used as a control well (BC).

When using Composition 2 medium, the prepared insulin-transferrin-selenium complex was added to a 48-well plate with inoculated cells of the primary culture, 250 μl per well so that the final concentrations of the initial solution of the insulin-transferrin-selenium complex were 1:200, 1:100 , 1:50 and 1:25, respectively (corresponding to final concentrations of insulin/transferrin/sodium selenite: 2.5 μg/ml, 1.25 μg/ml, 1.25 ng/ml; 5 μg/ml, 2.5 µg/ml, 2.5 ng/ml; 10 µg/ml, 5 µg/ml, 5 ng/ml; and 20 µg/ml, 10 µg/ml, 10 ng/ml, respectively); Composition 2 medium was used as a control well (BC).

When using Composition 3 medium, prepared epidermal growth factor was added to a 48-well plate with inoculated primary culture cells at a rate of 250 μl per well so that the final concentrations of epidermal growth factor were 100 ng/ml, 50 ng/ml/ml, 25 ng /ml and 12.5 ng/ml, respectively; Composition 3 medium was used as a control well (BC).

When using Composition 4 medium, prepared hepatocyte growth factor was added to a 48-well plate with inoculated primary culture cells at a rate of 250 μl per well so that the final concentrations of hepatocyte growth factor were 20 ng/ml, 10 ng/ml, 5 ng/ml and 2.5 ng/ml, respectively; Composition 4 medium was used as a control well (BC).

When using Composition 5 medium, prepared Y27632 was added at a rate of 250 μl per well to a 48-well plate with inoculated primary culture cells so that the final concentrations of Y27632 were 20 μM, 15 μM, 12.5 μM, 10 μM, 7.5 μM, 5 µM and 2.5 µM, respectively; Composition 5 medium was used as a control well (BC).

When using Composition 6 medium, prepared Compound 1 was added at a rate of 250 μl per well to a 48-well plate with inoculated primary culture cells so that the final concentrations of Compound 1 were 6 μM, 5 μM, 4 μM, 3 μM, 2 μM, 1. 5 µM and 0.75 µM, respectively; Composition 6 medium was used as a control well (BC).

When using Composition 7 medium, prepared A83-01 was added at a rate of 250 μl per well to a 48-well plate with inoculated primary culture cells so that the final concentrations of A83-01 were 1000 nM, 500 nM, 250 nM and 125 nM, respectively; Composition 7 medium was used as a control well (BC).

When using Formulation 8 medium, prepared SB202190 was added at a rate of 250 μl per well to a 48-well plate containing inoculated primary cells so that the final concentrations of SB202190 were 1000 nM, 500 nM, 250 nM, and 125 nM, respectively; Composition 8 medium was used as a control well (BC).

After the cells had grown to approximately 85% of the 48 wells, the cells were enzymatically disaggregated and counted relative to the number of cells in the control well (BC), and the results were shown in FIG. 3A-3N. In each of Fig. 3A-3H, the ratio is the ratio of the number of first subculture cells cultured using each culture medium to the number of first subculture cells cultured in the corresponding control well. If the ratio value is greater than 1, this indicates that the proliferation-stimulating effect of the prepared medium containing various concentrations of factors or low-molecular compounds is preferable to the effect of the control well medium; if the ratio value is less than 1, this indicates that the proliferation-stimulating effect of the prepared medium containing different concentrations of factors or low-molecular compounds is less than that of the control well medium.

According to the results shown in Fig. 3A-3H, the volume concentration of additive B27 in the culture medium is preferably 1:25-1:200, more preferably 1:25-1:50; the volume concentration of the insulin-transferrin-selenite complex is preferably 1:25-1:200, more preferably 1:25-1:100 (corresponding to final concentrations of insulin/transferrin/sodium selenite 2.5-20 μg/ml, 1.25- 10 µg/ml, 1.25-10 ng/ml, respectively, and more preferably 5-20 µg/ml, 2.5-10 µg/ml, 2.5-10 ng/ml, respectively); the amount of epidermal growth factor is preferably 12.5 ng/ml-100 ng/ml, more preferably 50 ng/ml-100 ng/ml; the amount of hepatocyte growth factor is preferably 2.5 ng/ml-20 ng/ml, more preferably 10 ng/ml-20 ng/ml; the amount of Y27632 is preferably 2.5 μM-15 μM and more preferably 7.5 μM-12.5 μM; the amount of Compound 1 is preferably 0.75 μM-6 μM, and more preferably 2 μM-6 μM; the amount of A83-01 is preferably 125-500 nM and more preferably 250-500 nM; the amount of SB202190 is preferably 125 nM to 500 nM, and more preferably 250 nM to 500 nM.

[Example 3]

Cultivation of primary human SCC epithelial cells (1) Cultivation of primary SCC tumor cells obtained from SCC tissue. Tumor tissue-derived RCC epithelial cells were isolated and prepared from the tumor tissue of an RCC patient (sample No. 0F0062) using the same method as in Example 1. Tumor tissue-derived RCC epithelial cells were then counted with using a flow cell imaging counter (JIMBIO FIL, Jiangsu Jimbio Technology Co., Ltd.) to obtain the total number of cells. Cells were then inoculated into a Matrigel® matrix-coated (purchased from BD Biosciences) 12-well plate at a density of 4 x 10 ⁴ cells/cm ² . 2 ml of prepared FEM culture medium for primary epithelial cells of SCRC was added to a 12-well plate, which was then placed in culture conditions at 37°C, 5% CO ₂ in an incubator (purchased from Thermo Fisher).

Fig. 4A is a microscopic image (photographed using a 40x inverted phase contrast microscope) of cells that were cultured until day 4 post-inoculation at a density of 4 x 10 ⁴ cells/cm ² in a 12-well plate coated with Matrigel matrix, in according to this Example. Microscopic observation shows that the cultured tumor cells of the primary culture of SCC, obtained from tumor tissue, were of high purity and did not contain fibroblasts. Fig. 4B is a microscopic image (photographed using a 40x inverted phase contrast microscope) of cells that were cultured until day 12 post-inoculation in accordance with the present Example. From Fig. 4A and 4B, it can be seen that the formation of an obvious clone can be observed under the microscope when the isolated cells of the primary culture of SCC were cultured in vitro for 4 days, and the number of cells increased significantly after 12 days of culture, which suggests that the technology of the present invention is an effective technology for growing PCR epithelial cells in vitro.

(2) Comparison of the results of culturing primary SCRC tumor cells obtained from SCRC tissue with and without coating with a gel-like extracellular matrix.

Tumor cells of the primary culture of SCC (No. 0F0065), obtained from tumor tissues, were isolated from the tumor tissues of one patient with SCC using the same method as in Example 1. Then equal quantities (4x10 ⁴ cells/cm ² ) of primary tumor 0F0065 PCR cells were inoculated into a 12-well plate coated with Matrigel® matrix (purchased from BD Biosciences) and into a 12-well plate without any treatment, respectively. 2 ml of prepared FEM culture medium for primary SCPC epithelial cells was added to 12-well plates, which were then placed in a 37°C, 5% CO ₂ incubator (purchased from Thermo Fisher) for culture after disinfection. Fig. 5A and 5B are microscopic images (photographed using a 100× inverted phase contrast microscope) of primary RCC 0F0065 tumor cells obtained from RCC tissue that were cultured until day 11 under Matrigel matrix-coated and non-Matrigel-coated conditions, respectively. From the figures it can be seen that the cell density and number of cells in Fig. 5A (with Matrigel matrix coating) is higher than in FIG. 5B (without coating with Matrigel matrix). At the same time, in FIG. 5B shows a small number of senescent cells (cells with larger cell bodies), whereas in FIG. 5And there is no cell aging. Therefore, it can be confirmed that the culture plate coated with Matrigel matrix is more favorable for the proliferation of SCC tumor cells than the culture plate without any treatment.

[Example 4]

The influence of various culture media on stimulating the proliferation of primary SCRC tumor cells obtained from SCRC tumor tissue

(1) Comparison of the effects of different culture media on the formation of primary culture cell clones and the effects of primary culture cell proliferation.

FEM culture medium for primary epithelial cells of PCRC was prepared in the same way as in Example 2, and BM basal medium was prepared as a control. As another control, FM culture medium was additionally prepared for the technology of condition-induced cell reprogramming. For preparation steps, see Liu et al., Nat Protoc, 12(2): 439–451, 2017. The composition of the culture medium is shown in Table 6. In parallel, CM culture medium was prepared for primary esophageal carcinoma cells as an additional control. For preparation steps, see Karin J. Purdie et al., Cancer Cell Culture: Methods and Protocols, Second Edition, Methods in Molecular Biology, vol. 731, 151-159, 2011. The composition of the culture medium is shown in Table 7. In addition, as an additional control, commercial EpiCult™ Plus Medium (hereinafter referred to as “EPI medium”) was purchased from Stemcell, and the composition of the culture medium is shown in Table 8.

Using the same method as in Example 1, tumor cells of the primary culture of SCC (No. 0F0060), the source of which were tumor tissues of SCC, were obtained. The cells were then cultured at the same density (4 x 10 ⁴ cells/cm ² ) under the following five culture conditions:

A. Invention technology: Primary culture tumor cells from SCLC were inoculated into a 24-well plate coated with Matrigel® (manufactured by BD Biosciences) at a density of 4 x 10 ⁴ cells/cm ² , cultured using 2 ml of the FEM culture medium of the present invention for primary epithelial cells. PCR cells;

B. Technology of condition-induced cell reprogramming: primary culture tumor cells from SCRC were inoculated onto a layer of mouse fibroblasts of the J2 cell line, treated with gamma rays (purchased from Kerafast) at a density of 4x10 ⁴ cells/cm ² , cultured in a 24-well plate with using FM medium for condition-induced cell reprogramming (for a detailed description of the steps, see Liu et al., Am J Pathol, 183(6): 1862-1870, 2013);

C. Primary culture tumor cells from SCC were inoculated into a Matrigel-coated 24-well plate® (manufactured by BD Biosciences) at a density of 4 x 10 ⁴ cells/cm ² , and cultured in the 24-well plate using 2 ml of KM culture medium;

D. Primary culture tumor cells from SCC were inoculated into a Matrigel-coated 24-well plate® (manufactured by BD Biosciences) at a density of 4 x 10 ⁴ cells/cm ² , and cultured in the 24-well plate using 2 ml of commercial EPI medium;

E. Primary culture tumor cells from SCC were inoculated into a Matrigel-coated 24-well plate® (manufactured by BD Biosciences) at a density of 4 x 10 ⁴ cells/cm ² and cultured in the 24-well plate using 2 ml of BM basic medium.

For the above five cultures, cells were grown under five culture conditions in which the media were refreshed every 4 days. At the same time, the cell clone formation and cell proliferation status were observed when cultured in each medium in a 24-well plate, and the cell growth status was recorded by photographing using a microscope (EVOS M500, Invitrogen).

As for the primary tumor cells of SCC (No. 0F0060), cultured using the technology of the invention, when the cells in the culture plate grew and covered about 80% of the bottom area, the supernatant of the medium was removed and 500 μl of 0.05% trypsin (purchased) was added to the 24-well plate from Gibco) for cell disaggregation; cells were incubated at 37°C for 10 minutes until complete cell disaggregation, which was observed under a microscope (EVOS M500, Invitrogen); then enzymatic disaggregation was stopped using 1 ml of DMEM/F12 culture solution containing 5% (v/v) fetal bovine serum, 100 U/ml penicillin and 100 μg/ml penicillin; the resulting mixture was collected in a 15 ml centrifuge tube and centrifuged at 1500 rpm for 4 minutes, and then the supernatant was removed. The centrifuged cell pellets were resuspended in the culture medium of the invention and counted using a flow cell imaging counter (JIMBIO FIL, Jiangsu Jimbio Technology Co., Ltd.) to obtain a total cell number of 364,000. Cells grown under the other four culture conditions , were enzymatically disaggregated and counted using the same operational process described above. The total number of cells cultured using FM, KM, EPI and BM media was 315,000, 91,000, 98,000 and 84,000, respectively.

Photos of cells in Fig. 6A-6E are microscopic photographs (under a 40× inverted phase contrast microscope) of sample #0F0060, which was cultured until day 14 under five different culture conditions: where FIG. 6A is a microscopic photograph of 0F0060 cultured until day 14 using BM basal medium; Fig. 6B is a microscopic photograph of 0F0060 cultured until day 14 using the FEM culture medium of the invention; Fig. 6C is a microscopic photograph of 0F0060 cultured until day 14 using KM culture medium; Fig. 6D is a microscopic photograph of 0F0060 cultured until day 14 using commercial EPI medium; and Fig. 6E is a microscopic photograph of 0F0060 cultured until day 14 using FM medium for condition-induced cell reprogramming. It is clear from the figures that sample 0F0060 could not form cell clones after 14 days of cultivation in basic VM medium (6A); when cultivated on KM (6C) and EPI (6D) culture media for 14 days, only a few cell clones with poor cellular status were formed; cells cultured in FM medium for induced cell reprogramming conditions (6E) for 14 days proliferated to a certain extent, but the cell density and number of cells were not comparable to those obtained in the FEM culture medium of the invention.

Fig. 7 is a comparative chart of the cell proliferation effects obtained by culturing primary cultured RCC cells isolated from tissue samples of six patients with RCC (No. 0F0056, 0F0060, 0F0061, 0F0062, 0F0071, 0F0075), according to Example 1, for 16 days of culture in conditions of five different culture media, where √ means moderate cloning ability and proliferation-promoting effect, √√ means significant cloning ability and proliferation-promoting effect, × means no cloning, and NT means “not tested.” From the image it follows that the culture medium according to the invention has a significant superiority over the other four cultivation conditions in terms of the ability to form clones and stimulating effect on cell proliferation when cultivating primary cultures from SCC tissues.

(2) Continuous cultivation and growth curve of tumor cells of primary cultures of SCC in various culture media

FEM culture medium for epithelial cells of primary PCR cultures and BM, KM, FM and EPI culture media used as controls were prepared using the same method as in (1) of this Example.

Primary culture RCC tumor cells derived from RCC tissue (No. 0F0075) were cultured in the following five culture media, and then subjected to enzymatic disaggregation, subculture and counting using the same method as in (1) of this Example.

Once the cells had grown in the culture plate after subculture and again covered about 80% of the bottom area of the plate, the cultured cells were enzymatically disaggregated, collected, and counted using the working method described above. Cells were again inoculated at a density of 4×10 ⁴ cells/well and continuously cultured.

The following is the formula for calculating the doubling of the cell population of primary epithelial cells of SCC under various culture conditions:

Population doubling (PD) = 3.32 * log ₁₀ (total number of cells disaggregated / number of cells at initial inoculation), see Chapman et al., Stem Cell Research & Therapy 2014, 5: 60.

In FIG. Figure 8 shows the growth curves of 0F0075 cells under five different culture conditions, plotted using Graphpad Prism software. The abscissa axis represents the days of cell culture, and the ordinate axis represents the multiple cumulative proliferation of cells, i.e. multiple expansion (reproduction) of cells during the cultivation period. The higher the value, the greater the expansion rate of the cell over a certain period, that is, the more cells multiply. The slope value represents the rate of cell proliferation. From the figure, it can be confirmed that the proliferation rate of SCRC epithelial cells cultured in the FEM culture medium according to the invention was higher than that of the other four culture conditions, and it can also be confirmed that the technology according to the invention can continuously culture primary SCRC epithelial cells, and the expansion rate remains unchanged after 30 days of breeding.

Photos of cells in Fig. 9A and 9C are microscopic photographs (under 40x inverted phase contrast microscope) of a primary culture of 0F0075 grown to first subculture under culture conditions in FEM and EPI media, respectively. Photos of cells in Fig. 9B and 9D are microscopic photographs (under 40x inverted phase contrast microscope) of a primary culture of 0F0075 grown to the ninth subculture under culture conditions in FEM and EPI media, respectively. From the figures it can be confirmed that the technology of the invention allows for the continuous cultivation of primary epithelial cells of SCRC, and the morphology of the cells after repeated subcultures and continuous cultivation has not changed significantly, compared with the morphology before subculture. However, the cell morphology changed significantly when the cells were cultured in commercial EPI medium until the 9th subculture, which could not meet the requirement of continuous culture of primary SCLC epithelial cells.

[Example 5]

Identification of primary PCR tumor cells derived from tumor tissues

(1) Immunohistochemical identification of primary SCRC tissues and primary SCRC culture cells after subculture

Tumor tissue (Specimen #0F0053) the size of a bean was taken from a surgical resection specimen from a patient with SCC and immersed in 1 ml of 4% paraformaldehyde. PCR epithelial cells (Sample No. 0F0053) were obtained from the remaining tumor tissue in the same way as in Example 1. Sample 0F0053 was continuously cultured until the sixth subculture using the FEM culture medium according to the invention, in accordance with the method of Example 3.

Immunohistochemical analysis was used to detect the expression of important biomarkers associated with SCC in the original tissue of sample 0F0053 and primary culture cells obtained by continuous culture until the sixth passage. The tissue was fixed with 4% paraformaldehyde, embedded in paraffin, and cut into 4-μm-thick sections using a microtome. Conventional immunohistochemical detection was then performed (for details of steps, see Li et al., Nature Communication, (2018) 9: 2983). The primary antibodies used were anti-cytokeratin (CK) (purchased from CST), anti-p63 (purchased from CST), anti-p53 (purchased from CST), and anti-Ki67 (purchased from R&D).

Fig. 10A and 10B confirm that the expression of SCC-related biomarkers on cells cultured from SCC tumor cells (Sample No. 0F0053) using the inventive technology up to the 6th passage was consistent with the expression of markers on the original tissue site from which the cells were obtained. This indicates that cells cultured using the technology of the invention retain the original pathological characteristics of tumor tissues of patients with PCR.

(2) Copy number analysis of SCRC tissues and SCRC cells disaggregated and cultured from tumor tissues.

RCC tumor cells (No. 0F0025) were continuously cultured using the FEM culture medium according to the invention, in accordance with the method of Example 3. For RCC tumor cells that were cultured and subcultured in vitro for the first, second and third time (P1, P2 and P3, respectively ), and tumor tissues obtained directly from patients with SCC through surgery, the genomic DNA of the cells was isolated using the DNeasy blood & tissue Kit, manufactured by QIAGEN. Normal esophageal tissues from the same patient were collected, and genomic DNA was extracted using the same method and used as a background control. Whole-exome sequencing of the genomic DNA of the cells and tissue samples was then performed (for specific steps, see Hans Clevers et al., Cell, 11; 172 (1-2): 373-386, 2018), and the sequencing results were compared with the reference genome. CNVkit software was used to analyze the comparison results. CBS segmentation (circular binary segmentation algorithm) was used to analyze the gene copy number of the whole genome, the features of regions with similar position and copy number changes were extracted using rolling median software, and the results were plotted according to chromosome order. Fig. 11A-11D confirm that the copy number changes in tumor tissue-derived SCC tumor cells that were cultured using the technology of the invention were substantially consistent with those in the patient's original tumor tissue.

[Example 6]

Effect of removal of one factor or a combination of factors from the culture medium on the continuous expansion of cells of primary SCRC culture FEM culture medium for primary epithelial cells of SCRC was prepared in the same way as in Example 2. As a control, basal medium (hereinafter also referred to as “BM”) was prepared ) using the same method as in Example 2. In addition, six other culture media were prepared according to Table 9.

Tissue-derived SCC primary culture tumor cells (#0F0061) were prepared using the same method as in Example 1. Primary SCC culture cells were then inoculated into a Matrigel® matrix-coated (manufactured by BD Biosciences) 24-well plate at a density of 4 x 10 ⁴ cells/ ^cm2 . 1 ml of the prepared FEM culture medium according to the invention was used, which was then placed in culture conditions at 37°C, 5% CO ₂ in an incubator (purchased from Thermo Fisher).

When the cells in the culture plate had grown to cover about 80% of the bottom surface area, the medium supernatant was removed and 400 μl of 0.05% trypsin (purchased from Gibco) was added to the 24-well plate to disaggregate the cells; cells were incubated at 37°C for 10 minutes until complete cell disaggregation, which was observed under a microscope (EVOS M500, Invitrogen); then enzymatic disaggregation was stopped using 800 μl of DMEM/F12 culture solution containing 5% (v/v) fetal bovine serum, 100 U/ml penicillin and 100 μg/ml penicillin; the resulting mixture was collected in a 15 ml centrifuge tube and centrifuged at 1500 rpm for 4 minutes, and then the supernatant was removed. The centrifuged cell pellets were resuspended in the culture medium of the invention and counted using a flow cell counter (JIMBIO FIL, Jiangsu Jimbio Technology Co., Ltd.) to obtain the total cell number. The cells were incubated in another 24-well culture plate coated with a gel-like extracellular matrix at a density of 2×10 ⁴ cells/cm ² for further cultivation. Cells were inoculated into a 24-well culture plate coated with gelled extracellular matrix at the same density using another seven different culture media.

Once the cells had grown in the culture plate after subculture and again covered about 80% of the bottom area of the plate, the cultured cells were enzymatically disaggregated, collected, and counted using the working method described above. The cells were again inoculated at a density of 2×10 ⁴ cells/cm ² and continuously cultured using appropriate culture media of various compositions.

The following is the formula for calculating the doubling of the cell population of primary epithelial cells of SCC under various culture conditions:

Population doubling (PD) = 3.32 * log ₁₀ (total number of cells disaggregated / number of cells at initial inoculation), see Chapman et al., Stem Cell Research & Therapy 2014, 5:60.

In FIG. Figure 12 shows cell growth curves under eight different culture conditions, plotted using Graphpad Prism software. The abscissa axis represents the days of cell culture, and the ordinate axis represents the multiple cumulative proliferation of cells, i.e. multiple expansion of cells during the cultivation period. The higher the value, the greater the expansion rate of the cell over a certain period, that is, the more cells multiply. The slope value represents the rate of cell proliferation.

Photos of cells in Fig. 13A-13H are microscopic photographs (under 100× inverted phase contrast microscope) of 0F0061 cultured to the third subculture under the above eight different culture conditions.

As can be seen from those shown in Fig. 12 and Fig. 13 results, the effect of cell proliferation was significantly weakened after removing Compound 1, Y27632, A83-01 or SB202190 alone, or the combination of Compound 1 and Y27632, or the combination of A83-01 and SB202190, from the culture medium of the invention. Additionally, after removal of the combination components of Compound 1 and Y27632, the cell proliferation rate was significantly reduced, suggesting that the combination components of Compound 1 and Y27632 in the culture medium of the invention are necessary for cell proliferation and continuous culture.

[Example 7]

Experiments on a xenograft model of tumorigenesis in mice using primary cultured SCRC cells obtained from tumor tissue

RCC tumor cells (No. 0F0056) were isolated and prepared from tumor tissues of one patient pathologically diagnosed with RCC using the same method as in Example 1. 0F0065 cells were cultured using the FEM culture medium of the invention according to the method of Example 3, and when the number of tumor cells PCR cells reached 1×10 ⁷ , PCR tumor cells were disaggregated and collected using the same method as in Example 4. FEM culture medium for PCR tumor cells according to the invention and Matrigel® matrix (purchased from BD Biosciences) were mixed in a 1:1 ratio , and 100 μl of culture medium mixed with Matrigel was used to resuspend 5×10 ⁶ SCC tumor cells, and the resulting solution was injected into the SCC fat pad and axilla of the right forelimb of 6-week-old female highly immunocompromised (NCG-) mice. mice) (purchased from Nanjing Model Animal Research Center), respectively. The volume and growth rate of tumors in mice derived from SCC tumor cells were observed and photographed every three days.

Tumor formation can be observed at both of the two tumor cell inoculation sites in mice on day 15 after tumor cell inoculation. From days 15 to 30, tumor proliferation in mice was obvious. This indicates that SCC tumor cells derived from tumor tissue cultured by the culture method according to the invention are tumorigenic in mice.

[Example 8]

Functional drug sensitivity test of PCR tumor cells derived from tumor tissue

Taking a surgical resection sample from a patient with RCC as an example, it can be seen that RCC tumor cells cultured from patient-derived RCC tumor samples can be used to test the sensitivity of the patient's tumor cells to various drugs.

1. Seeding of Primary SSCC Tumor Cells: Cell suspensions of isolated SSCC tumor cells (No. 0F0060 and No. 0F0062) obtained in accordance with the method of Example 1 were inoculated into a Matrigel matrix-coated 12-well® plate (purchased from BD Biosciences) in density 4×10 ⁴ cells/cm ² . 2 ml of prepared FEM culture medium for primary epithelial cells was added to a 12-well plate, which was then placed in culture conditions at 37°C, 5% CO ₂ (purchased from Thermo Fisher). When the cells in the culture plate had grown to cover about 80% of the bottom surface area, the medium supernatant was removed and 500 μl of 0.05% trypsin (purchased from Gibco) was added to the 12-well plate to disaggregate the cells; cells were incubated at 37°C for 10 minutes until complete cell disaggregation, which was observed under a microscope (EVOS M500, Invitrogen); then enzymatic disaggregation was stopped using 1 ml of DMEM/F12 culture solution containing 5% (v/v) fetal bovine serum, 100 U/ml penicillin and 100 μg/ml penicillin; the resulting mixture was collected in a 15 ml centrifuge tube and centrifuged at 1500 rpm for 4 minutes, and then the supernatant was removed. The centrifuged cell pellets were resuspended in FEM culture medium and counted using a flow cell imaging counter (JIMBIO FIL, Jiangsu Jimbio Technology Co., Ltd.) to obtain the total cell numbers, which were 830,000 and 768,000, respectively. Cells were incubated in a 384-well plate at a density of 2000-4000 cells/well, and cells adhered to the surface overnight.

2. Drug gradient experiments:

(1) Drug storage plates were prepared by gradient dilution method: 10 μL of the drug stock solutions to be tested (the concentration of the drug stock solution was determined based on 2 times the maximum blood concentration Cmax in the human body), respectively, was withdrawn and added to the EP 0.5 ml tubes containing 20 µl DMSO; 10 μl of solutions from the above EP tubes were pipetted into second EP tubes with a volume of 0.5 ml, with 20 μl of DMSO previously added, that is, the drugs were diluted in a ratio of 1:3. The above method was repeated for gradual dilution to obtain the 7 concentrations required for dosing. Various drug concentrations were added to 384-well drug storage plates. 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 bortezomib (purchased from MCE), mitomycin (purchased from MCE), hydroxycamptothecin (purchased from MCE), and erlotinib (purchased from MCE).

(2) Using a high-throughput automated workstation (JANUS, Perkin Elmer), various concentrations of drugs and solvent controls in 384-well drug storage plates were added to 384-well cell culture plates containing SCC tumor cells. The drug and vehicle control groups each had 3 duplicate wells. The volume of drugs added to each well was 100 nl.

(3) Cell viability test: 72 hours after drug administration, a Cell Titer-Glo kit (purchased from Promega) was used to determine the amount of chemiluminescence of cultured cells after drug administration. 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 after mixing, a microplate reader (Envision, Perkin Elmer) was used to determine the chemiluminescence value.

(4) Cell viability test: According to the formula: Cell viability (%) = drug well chemiluminescence value / control well chemiluminescence value * 100%, the survival rates of cells treated with various drugs were calculated. Graphs were made using Graphpad Prism software and IC ₅₀ values were calculated.

(5) The results of drug sensitivity testing are shown in FIG. 14.

Fig. 14A and 14B, respectively, show the sensitivity of RCC tumor cells cultured from surgically resected tumor tissue samples from two different RCC patients (Sample #0F0060 and Sample #0F0062) to the two chemotherapeutic drugs mitomycin and hydroxycamptothecin, as well as targeted drugs. effects of bortezomib and erlotinib. In particular, in FIG. 14A shows the sensitivity results of PCR cells cultured from sample No. 0F0060 to four drugs; in Fig. 14B shows the sensitivity results of PCR cells cultured from sample No. 0F0062 to four drugs. The results show 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.

Industrial applicability

According to the invention, a culture medium and a culture method are proposed for growing or propagating primary epithelial cells of SCC in vitro. Cultured cells can be used to evaluate efficacy and screen drugs. Therefore, the invention is applicable in industry.

Although the present invention has been described in detail herein, the present invention is not limited thereto, and modifications may be made by those skilled in the art in accordance with the principle of the invention. Therefore, all modifications made in accordance with the principle of the invention should be considered as falling within the scope of protection of the invention.

Claims (54)

1. A primary cell culture medium for culturing primary epithelial cells of esophageal squamous cell carcinoma (ESCC), characterized in that: includes an MST1/2 kinase inhibitor and a ROCK kinase inhibitor, wherein the MST1/2 kinase inhibitor comprises a compound of Formula (I) or a pharmaceutically acceptable salt or solvate thereof, and the ROCK kinase inhibitor is at least one selected from the group consisting of Y27632 , Fasudila and N-1152, Formula (I) Where R ₁ is selected from C1-C6 alkyl, C3-C6 cycloalkyl, C4-C8 cycloalkylalkyl, C2-C6 spirocycloalkyl and aryl, optionally independently containing 1-2 R ₆ as substituents, aryl-C1-C6 alkyl, optionally independently containing as substituents 1-2 R ₆ , and heteroaryl, optionally independently containing 1-2 R ₆ as substituents; 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, hydroxyl-C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkylamino-C1-C6 alkyl, C1-C6 alkoxy-C1-C6 alkyl and C3-C6 heterocyclyl-C1-C6 alkyl; R ₆ is selected from halogen, C1-C6 alkyl, C1-C6 alkoxy and C1-C6 haloalkyl. 2. Primary cell culture medium according to claim 1, characterized in that R ₁ is selected from C1-C6 alkyl, C3-C6 cycloalkyl, C4-C8 cycloalkylalkyl, C2-C6 spirocycloalkyl and phenyl, optionally independently containing 1-2 R ₆ as substituents, naphthyl, optionally independently containing 1-2 R as substituents ₆ , phenylmethyl, optionally independently containing 1-2 R ₆ as substituents, and thienyl, optionally independently containing 1-2 R ₆ as substituents; 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, C1-C6 alkyl hydroxyl, C1-C6 haloalkyl, C1-C6 alkylamino, C1-C6 alkyl, C1 -C6 alkoxy C1-C6 alkyl, piperidyl C1-C6 alkyl and tetrahydropyranyl C1-C6 alkyl; R ₆ is selected from halogen, C1-C6 alkyl, C1-C6 alkoxy and C1-C6 haloalkyl. 3. Primary cell culture medium according to claim 1, characterized in that the MST1/2 kinase inhibitor contains a compound of formula (Ia) or a pharmaceutically acceptable salt or solvate thereof, Formula (Ia) Where R ₁ is selected from C1-C6 alkyl, phenyl, optionally independently containing 1-2 R ₆ as substituents, thienyl, optionally independently containing 1-2 R ₆ as substituents, and phenylmethyl, optionally independently containing 1-2 R as substituents ₆ ; R ₅ is selected from hydrogen, C1-C6 alkyl and C3-C6 cycloalkyl; R ₆ is independently selected from halogen, C1-C6 alkyl and C1-C6 haloalkyl. 4. Primary cell culture medium according to claim 3, characterized in that R ₁ is phenyl, optionally independently substituted with 1-2 R ₆ ; R ₅ represents hydrogen; R ₆ is preferably fluorine, methyl or trifluoromethyl. 5. Primary cell culture medium according to claim 1, characterized in that the MST1/2 kinase inhibitor is at least one compound selected from the following compounds, or a pharmaceutically acceptable salt thereof: 1 2 3 4 5 6 7 8 9 10 eleven 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 thirty 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 . 6. Primary cell culture medium according to any one of paragraphs. 1-5, characterized by the fact that: the amount of MST1/2 kinase inhibitor is 0.3 μM-10 μM, preferably 0.75 μM-6 μM, more preferably 2 μM-6 μM. 7. Primary cell culture medium according to any one of paragraphs. 1-5, characterized by the fact that: the amount of ROCK kinase inhibitor in the culture medium is 0.3 μM to 20 μM, preferably 2.5 μM to 15 μM, more preferably 7.5 μM to 12.5 μM. 8. Primary cell culture medium according to any one of paragraphs. 1-5, characterized by the fact that: it further contains one or more of the following factors: epidermal growth factor; insulin-transferrin-selenium complex; additive B27 or additive N2; hepatocyte growth factor; a TGFβ type I receptor inhibitor selected from at least one of A83-01, SB431542, Repsox, SB505124, SB525334, SD208, LY36494 and SJN2511; and a P38/MAPK inhibitor selected from at least one of SB202190, SB203580, VX-702, VX-745, PD169316, RO4402247, and BIRB796. 9. Primary cell culture medium according to claim 8, characterized in that: the amount of epidermal growth factor is 12.5 ng/ml-100 ng/ml, more preferably 50 ng/ml-100 ng/ml; the corresponding amounts of insulin/transferrin/sodium selenite in the insulin-transferrin-selenium complex are 2.5-20 μg/ml, 1.25-10 μg/ml, 1.25-10 ng/ml, respectively, and more preferably 5- 20 µg/ml, 2.5-10 µg/ml, 2.5-10 ng/ml, respectively; the volume concentration of additive B27 or additive N2 is 1:25-1:200, more preferably 1:25-1:50; the amount of hepatocyte growth factor is 2.5 ng/ml-20 ng/ml, more preferably 10 ng/ml-20 ng/ml; the amount of TGFβ type I receptor inhibitor is 125-500 nM, preferably 250-500 nM; and the amount of P38/MAPK inhibitor is 125-500 nM, preferably 250-500 nM. 10. Primary cell culture medium according to any one of paragraphs. 1-5, characterized by the fact that: does not contain whey, bovine pituitary extract, Wnt agonists, R-spondin family proteins, BMP inhibitors, nicotinamide or N-acetylcysteine. 11. Primary cell culture medium according to any one of paragraphs. 1-5, characterized by the fact that: the epithelial cells of the primary cultures of PCR are selected from tumor cells of PCR, normal epithelial cells of PCR and epithelial stem cells of PCR. 12. A method for cultivating epithelial cells of primary PCR cultures, characterized in that it includes the following steps: (1) preparing a primary cell culture medium according to any one of paragraphs. 1-11; (2) coating the culture vessel with a diluted gel-like extracellular matrix; (3) inoculating primary SCPC epithelial cells isolated from SCPC tissues into a coated culture vessel and culturing using the primary cell culture medium from step (1). 13. A method for assessing the effectiveness of a drug candidate for the treatment of SCC diseases, characterized by the fact that it includes the following steps: (1) culturing epithelial cells of SCRC by the culturing method according to claim 12; (2) selection of the candidate drug to be tested and dilution in different drug concentration gradients; (3) adding the specified drug candidate, diluted to gradients, to the SCC epithelial cells obtained in step (1), and determining the viability of the cells. 14. A method for screening a drug candidate for the treatment of SCC diseases, characterized in that it includes the following steps: (1) culturing epithelial cells of SCRC by the culturing method according to claim 12; (2) selection of the candidate drug substance to be tested and dilution into different drug concentration gradients; (3) adding the drug candidate, diluted to gradients, to the SCRC epithelial cells obtained in step (1) and determining cell viability.