CN113969262A

CN113969262A - Culture medium for lung cancer epithelial cells, culture method and application thereof

Info

Publication number: CN113969262A
Application number: CN202010708892.6A
Authority: CN
Inventors: 刘青松; 胡洁; 王文超; 陈程; 任涛; 王黎
Original assignee: Precedo Pharmaceuticals Co Ltd
Current assignee: Precedo Pharmaceuticals Co Ltd
Priority date: 2020-07-22
Filing date: 2020-07-22
Publication date: 2022-01-25
Anticipated expiration: 2040-07-22
Also published as: WO2022016642A1; CN113969262B

Abstract

The invention provides a primary cell culture medium for culturing lung cancer epithelial cells, which contains an MST1/2 kinase inhibitor; insulin-like growth factor 1; an epidermal growth factor; hepatocyte growth factor; neuregulin-1; an additive selected from at least one of insulin-transferrin-selenium complex, B27 additive, and N2 additive; a ROCK kinase inhibitor selected from at least one of Y27632, fasudil, and H-1152. The invention also provides a culture method using the primary cell culture medium, and a method and application of the culture medium and the culture method for evaluating and screening the curative effect of a medicament.

Description

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

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to a culture medium and a culture method for in vitro culture or amplification of primary lung cancer epithelial cells, and a method and application of cultured cells in drug efficacy evaluation and screening.

Background

Lung cancer is still one of the most common and lethal tumors, with 160 million deaths associated with tumors worldwide each year. Lung cancer is a diverse, complex, and therapeutically challenging disease. Over the past decade, with the advent of personalized medicine, further understanding of the underlying biology and molecular mechanisms of lung cancer has emerged. Lung cancer is no longer a single disease entity and is now subdivided into molecular subtypes, each corresponding to a specialized targeted chemotherapeutic strategy.

Functional testing refers to the in vitro detection of the sensitivity of anti-tumor drugs on cells of cancer patients. The key to applying this method is to develop a tumor cell model with a short growth cycle and capable of representing the own biological characteristics of the lung cancer patient. In addition, the cell model is convenient to operate and can quickly and efficiently predict the curative effect of clinical medication, so that accurate medication guidance is given to cancer patients in time. However, the success rate of establishing a cell model in vitro by using primary tumor cells from cancer patients is often low, the growth cycle is long, and the problems of excessive proliferation of mesenchymal cells such as fibroblasts exist, which restricts the development of the field. Currently, two techniques for culturing primary epithelial cells/stem cells have been developed relatively well in the field of tumor cell functionality test applications, one is a technique that uses irradiated trophoblasts and ROCK kinase inhibitors to promote the growth of primary epithelial cells to examine drug sensitivity of individual patients, i.e., a cell conditioning reprogramming technique (Liu et al, am.j.pathol., 180: 599-. Another technique is the in vitro 3D culture of adult stem cells to obtain organoid techniques similar to tissues and organs (Hans Clevers et al, Cell, 11, 172 (1-2): 373-386, 2018).

However, organoid technology is a technology in which patient autologous primary epithelial cells are embedded in extracellular matrix for in vitro three-dimensional culture, but the culture medium of the technology needs to be added with a plurality of specific growth factors (such as Wnt proteins and R-spondin family proteins), which is expensive and not suitable for clinical large-scale application. In addition, cells need to be embedded in extracellular matrix gel in the whole culture process of the organoid, the plating steps of cell inoculation, passage and drug sensitivity test are complicated and time-consuming compared with the 2D culture operation, the size of the organoid formed by the technology is not easy to control, and the situation that the inside of the organoid is necrotized due to the fact that the part of the organoid grows too large is easy to occur. Therefore, organoid techniques are less operable and adaptable than 2D culture techniques, require the operation of a skilled technician, and are not suitable for wide application on a large scale in clinical in vitro drug sensitivity assays (Nick Barker, nat. cell biol., 18 (3): 246-54, 2016).

The cell reprogramming technology is a technology for co-culturing autologous primary epithelial cells of a patient and murine feeder cells, a culture medium for in vitro amplification of the primary lung cancer cells in the existing literature contains serum components, and the serum components are undefined, so that the difference between different batches is large, and certain interference is easily generated on an experimental result.

In view of the limitations of the above technologies, there is a clinical need to develop a primary lung cancer epithelial cell culture technology, which has a short culture period, controllable cost and convenient operation, and when the technology is applied to construct a primary lung cancer tumor cell model, the cultured lung cancer tumor cells can represent the biological characteristics of a lung cancer patient. By evaluating the sensitivity of the antitumor drug on cell models derived from different cancer patients in vitro, the response rate of the antitumor drug in clinic is improved, and the pain of the patients and the waste of medical resources caused by inappropriate drugs are reduced.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides a culture medium for culturing primary lung cancer epithelial cells and a culture method of the primary lung cancer epithelial cells by using the culture medium. By adopting the primary lung cancer epithelial cell culture medium and the culture method, the purposes of short in-vitro culture period, controllable cost and convenient and fast operation can be achieved. When the technology is applied to the construction of a primary lung cancer tumor cell model, the primary lung cancer tumor cells with the own biological characteristics of a lung cancer patient can be obtained, and the technology can be applied to new drug screening and in-vitro drug sensitivity detection.

One aspect of the invention is to provide a primary cell culture medium for culturing primary lung cancer epithelial cells, comprising an MST1/2 kinase inhibitor; insulin-like growth factor 1 (IGF-1); epidermal Growth Factor (EGF); hepatocyte Growth Factor (HGF); neuregulin-1; an additive selected from at least one of insulin-transferrin-selenium complex (ITS), B27 additive, and N2 additive; a ROCK kinase inhibitor selected from at least one of Y27632, fasudil, and H-1152, said MST1/2 kinase inhibitor comprising a compound of formula (I) or a pharmaceutically acceptable salt, or solvate thereof.

Wherein the content of the first and second substances,

R₁selected from C1-C6 alkyl, C3-C6 cycloalkyl, C4-C8 cycloalkylalkyl, C2-C6 spirocycloalkyl, and optionally substituted with 1-2 independent R₆Substituted aryl (e.g., phenyl, naphthyl, and the like), aryl C1-C6 alkyl (e.g., benzyl, and the like), and heteroaryl (e.g., thienyl, and the like);

R₂and R₃Each independently selected from C1-C6 alkyl, preferably C1-C3 alkyl, more preferably methyl;

R₄and R₅Each independently selected from hydrogen, C1-C6 alkyl, C3-C6 cycloalkyl, C4-C8 cycloalkylalkyl, C1-C6 alkylhydroxy, C1-C6 haloalkyl, C1-C6 alkylamino C1-C6 alkyl, C1-C6 alkoxy C1-C6 alkyl, and C3-C6 heterocyclyl C1-C6 alkyl (said heterocyclyl is selected from, for example, piperidinyl, tetrahydropyranyl, etc.);

R₆selected 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, the MST1/2 kinase inhibitor comprises a compound of formula (Ia) or a pharmaceutically acceptable salt, or solvate thereof,

wherein the content of the first and second substances,

R₁selected from C1-C6 alkyl, optionally substituted with 1-2 independent R₆Substituted phenyl, optionally substituted with 1-2 independent R₆Substituted thienyl, and optionally substituted with 1-2 independent R₆Substituted benzyl, R₁More preferably optionally substituted with 1-2 independent R₆Substituted phenyl;

R₅selected from hydrogen, C1-C6 alkyl, and C3-C6 cycloalkyl, R₅More preferably hydrogen;

R₆each independently selected from halogen, C1-C6 alkyl, and C1-C6 haloalkyl, R₆More preferably fluorine, methyl or trifluoromethyl.

Preferably, the MST1/2 inhibitor is at least one 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.

In an embodiment of the invention, the amount of MST1/2 kinase inhibitor in the culture medium is typically in the range of 0.625 μ M to 20 μ M, preferably 0.625 μ M to 10 μ M, more preferably 2.5 μ M.

In a preferred embodiment, the content of insulin-like growth factor 1 is preferably 1.25-80 ng/ml, more preferably 5-80 ng/ml; the content of the hepatocyte growth factor is preferably 5-80 ng/ml, and more preferably 20-80 ng/ml; the content of the epidermal growth factor is preferably 10-80 ng/ml, and more preferably 10-40 ng/ml; the content of the neuregulin-1 is preferably 10-80 ng/ml, and more preferably 20-80 ng/ml; the additive is preferably an insulin-transferrin-selenium complex, wherein the content of the insulin/transferrin/sodium selenite is preferably 2.5-20 mug/ml-1.25-10 ng/ml, and the content of the insulin/transferrin/sodium selenite is preferably 10-20 mug/ml-5-10 ng/ml; the content of the ROCK kinase inhibitor is preferably 1.25-20 mu M, more preferably 2.5-10 mu M, and the ROCK kinase inhibitor is preferably Y27632.

Compared with the components of a cell condition reprogramming culture medium and an organoid culture medium, the components of the culture medium formula are added with an MST1/2 kinase inhibitor, but the culture medium formula does not contain uncertain components such as serum, bovine pituitary extract and the like, does not contain niche factors necessary for culturing organoids such as Wnt agonist, R-spondin family protein, BMP inhibitor and the like, and does not contain nicotinamide and N-acetylcysteine, so that the cost of the culture medium is greatly reduced, the operation flow for preparing the culture medium is simplified, and the in-vitro culture of primary lung cancer epithelial cells with controllable cost and convenient operation is realized.

In the invention, the primary lung cancer epithelial cells can be lung cancer tumor cells, normal lung cancer epithelial cells and lung cancer epithelial stem cells.

One aspect of the present invention provides a method for culturing primary lung cancer epithelial cells, comprising the following steps:

(1) the primary cell culture medium of the invention is prepared according to the formula.

(2) Pre-plating the culture vessel with irradiated feeder cells.

Specifically, the feeder cells can be irradiated NIH-3T3 cells, the irradiation source is X rays or gamma rays, preferably gamma rays, and the irradiation dose is 30-50 Gy, preferably 35 Gy. Specifically, irradiated NIH-3T3 cells were treated at 2X 10⁴Per cm²And (3) inoculating the cells in a culture container such as a 48-well plate, a 24-well plate, a 12-well plate, a 6-well plate or a T25 cell culture bottle, and reserving the cells after the cells are attached.

(3) Separating lung cancer tissue to obtain primary lung cancer epithelial cells.

Primary lung cancer epithelial cells can be derived, for example, from lung cancer tissue samples and lung cancer punctures or lung endobronchial endoscopic samples. The lung cancer tissue sample is derived from, for example, a cancer tissue sample surgically removed from a patient having an indicated and consented lung cancer tumor, and the lung cancer puncture or endobronchial sample is derived from, for example, a puncture or endobronchial sample taken while the patient having an indicated and consented lung cancer tumor is being biopsied. The collection of the tissue sample is performed within half an hour after surgical resection or biopsy of the patient. More specifically, a tissue sample of a non-necrotic area is taken in a sterile environment, and the volume of the tissue sample is 0.5cm³Placing the culture medium into a precooled DMEM/F12 culture medium of 3-5mL, placing the culture medium in a plastic sterile centrifuge tube with a cover, and transporting the culture medium to a laboratory on ice; wherein the DMEM/F12 medium contains 1-2 vol% of penicillin/streptomycin and/or 0.2-0.4 vol% of Primocin (hereinafter referred to as tissue transfusion solution). When streptomycin/penicillin is used, the concentration range of the streptomycin is 25-400 mu g/mL, preferably 50-200 mu g/mL, more preferably 200 mu g/mL, and the concentration range of the penicillin is 25-400U/mL, preferably 50-200U/mL, more preferably 200U/mL; when Primocin is used, the concentration is in the range of 25 to 400. mu.g/mL, preferably 50 to 200. mu.g/mL, more preferably 100. mu.g/mL.

In the biological safety cabinet, the tissue sample is transferred to a cell culture dish, the tissue sample is rinsed by using a transport solution, and blood cells on the surface of the tissue sample are cleaned. Transferring the tissue sample after being moistened into another new culture dish, adding 1-3mL of transport solution, and cutting the tissue sample into pieces with the volume less than 3mm by using a sterile surgical blade and surgical forceps³The tissue fragment of (a).

Transferring the tissue sample fragments into a centrifuge tube, and centrifuging for 3-5 minutes at 1000-3000 r/min by using a desktop centrifuge (3-18K of Sigma company); discarding the supernatant, adding tissue transfusion liquid and tissue digestive fluid (5 mL of tissue digestive fluid is used per 10mg of tissue according to the ratio of 1:1, wherein the preparation method of the tissue digestive fluid comprises the steps of dissolving 1-2 mg/mL collagenase II, 1-2 mg/mL collagenase IV, 50-100U/mL deoxyribonucleic acid I, 0.5-1 mg/mL hyaluronidase, 0.1-0.5 mg/mL calcium chloride and 5-10 mg/mL bovine serum albumin in HBSS and RPMI-1640 with the volume ratio of 1: 1), marking the sample number, sealing a sealing film, digesting by a constant temperature shaking table (known as ZQLY-180N) with the rotation speed of 200-300 at 37 ℃, and observing whether the digestion is finished or not at intervals of 1 hour; if no obvious tissue block is found, the digestion can be stopped, otherwise, the digestion is continued until the digestion is sufficient, and the digestion time range is 4-8 hours. After digestion, the undigested tissue mass is filtered off by a cell strainer (the cell mesh size is, for example, 70 μm), the tissue mass on the strainer is washed with a tissue transport fluid, the residual cells are washed into a centrifuge tube, and the centrifuge tube is centrifuged at 1000 to 3000 rpm for 3 to 5 minutes by a desk centrifuge. Discarding the supernatant, observing whether the residual cell mass contains blood cells, adding 1-5 mL of blood cell lysate (purchased from Sigma company) if the blood cells exist, uniformly mixing, carrying out lysis at 4 ℃ for 10-20 minutes, shaking for 5 minutes, uniformly mixing once, taking out after the lysis is finished, and centrifuging for 3-5 minutes at 1000-3000 r/min. The supernatant was discarded, the primary cell culture medium of the present invention was added to resuspend the cells, and the total number of cells was counted using a flow cytometer (jiamboo FIL, jiangsu microbial technology ltd).

(4) Inoculating the primary lung cancer epithelial cells separated in the step (3) into a culture vessel pre-inoculated with the trophoblasts, and culturing by adopting the primary cell culture medium in the step (1).

More specifically, the ratio of 2X 10 in one well of the multi-well plate is preliminarily set⁴～4×10⁴Per cm²(e.g., 2X 10)⁴Per cm²) The NIH-3H3 cells after 35Gy radiation dose gamma ray radiation are inoculated at the density, and after the cells adhere to the wall, the cell density is 2 multiplied by 10⁴～8×10⁴Per cm²(e.g., 4X 10)⁴Per cm²) Inoculating primary lung cancer tumor cells at a density of 0.5-2 mL per well of primary epithelial cell culture medium at, e.g., 37 ℃ and 5% CO₂Culturing in a cell culture box for 8-16 days, changing into a fresh primary cell culture medium every 4 days, and performing digestion passage when the primary lung cancer epithelial cells grow to the cell density of about 80-90% of the bottom area of the porous plate.

Compared with organoid technology, the step does not need to mix the primary cells and the matrigel uniformly on ice to form gel drops, and adds the culture medium after the gel drops are solidified. In addition, the use amount of the expensive extracellular matrix glue is saved, and the operation steps are simplified.

Optionally, after the inoculated primary lung cancer epithelial cells are cultured for 8-16 days, when cell clones formed in a culture container are converged to reach a basal area of 80%, discarding the supernatant, adding 1-2 mL of 0.25% pancreatin (purchased from Thermo Fisher company) for digestion for 1 minute, then sucking out 0.25% pancreatin, adding 1-2 mL of 0.05% pancreatin for cell digestion, and incubating for 5-20 minutes at room temperature; then, 2 to 4mL of a culture medium containing, for example, 5% (v/v) fetal bovine serum, 100U/mL penicillin and 100. mu.g/mL streptomycin is used to resuspend the digested cells, centrifuged at 1000 to 3000 rpm for 3 to 5 minutes, the digested single cells are resuspended in the primary cell culture medium of the present invention, and the resulting cell suspension is placed in a T25 cell culture flask pre-plated with feeder cells to continue the expansion culture. The pretreatment operation of the T25 cell culture flask is the same as that in the step (2).

The amplified lung cancer epithelial cells grow in 2D, and the conditions that the organoid technology is amplified to cause the size of the organoid to be uneven and the organoid with over-growth to cause the internal necrosis and the like are avoided.

On the other hand, the lung cancer epithelial cells, particularly lung cancer tumor cells, cultured by the method for culturing primary lung cancer epithelial cells of the present invention can be used for evaluating and screening the curative effect of a medicament, and the method comprises the following steps:

(1) obtaining primary lung cancer epithelial cells, particularly preferably a cancer tissue sample or biopsy derived from a patient with lung cancer, isolating the primary lung cancer epithelial cells, culturing and expanding the primary lung cancer epithelial cells (particularly primary lung cancer tumor cells) to at least 10% according to the primary lung cancer epithelial cell culture method described above⁵Of order of magnitude, preferably at least 10⁶An order of magnitude of cell number.

(2) The drug to be tested is selected.

(3) The drug at its maximum plasma concentration C_maxFor reference, 2-5 times C_maxFor the starting concentration, a plurality of different drug concentration gradients is diluted, for example 5-10, preferably 6-8 drug concentration gradients.

(4) Digesting the lung cancer epithelial cells obtained by culturing in the step (1) into a single cell suspension, counting by using a flow image counter, diluting the single cell suspension by using the primary cell culture medium of the invention, uniformly adding the diluted cell suspension into a multi-well plate according to the density of 2000-4000 cells per well, for example, 50 mu L of cell diluent per well, and carrying out overnight adherence.

The step avoids the problem that the cell reprogramming technology interferes with primary cell counting and subsequent primary cell activity detection due to the existence of feeder cells, and the complicated step of mixing, embedding and then plating cell suspension and matrigel on ice like the organoid technology is not needed, so that the operation flow is greatly simplified, and the operability and the practicability of the technology are enhanced. Because the inoculated cells are single cell suspension instead of 3D structure like organoid, compared with organoid technology, the technology has the advantages that the number of the plated cells is more uniform, the difference of the number of the cells among the holes generated by plating is small, and the technology is more suitable for subsequent high-throughput drug screening operation.

(5) And (4) adding selected candidate drugs such as traditional chemotherapeutic drugs, targeted drugs, antibody drugs or a combination of a plurality of drugs and the like after gradient dilution to the adherent cells obtained in the step (4) by adopting a high-throughput automatic workstation for processing.

(6) After several hours, for example, 72 hours after the drug treatment, the survival rate of lung cancer epithelial cells is examined using a Cell-Titer Glo luminescence Cell viability assay kit (purchased from Promega corporation) and drug activity screening is performed.

Specifically, for example, 10. mu.L of Cell Titer-Glo reagent (available from Promega corporation) was added to each well, and after uniform shaking, the chemiluminescence intensity of each well was measured with a fluorescence microplate reader, and the inhibition intensity of each drug against the proliferation of the test cells was calculated from the measured values by plotting the drug concentration on the abscissa and the fluorescence intensity on the ordinate using GraphPad Prism software.

The primary lung cancer tumor cell is applied to drug screening and in-vitro drug sensitivity detection. Since cells grow in 2D, the duration of action with the drug is also shorter than the drug detection time of organoid technology (mean administration time of organoid technology is 6 days).

The beneficial effects of the invention also include:

(1) the success rate of primary lung cancer epithelial cell culture is improved and reaches more than 85 percent;

(2) ensuring that the lung cancer epithelial cells subjected to in vitro primary culture can maintain the pathological phenotype and heterogeneity of patients with primary cell sources;

(3) the components of the culture medium do not contain serum, so the culture medium is not influenced by the quality and quantity of serum of different batches;

(4) the efficiency of expanding lung cancer epithelial cells is high, only 10⁴The cell number of the grade can be successfully amplified to 10 within about two weeks⁶The lung cancer epithelial cells of the magnitude order can be amplified, and the amplified lung cancer epithelial cells can be continuously passaged;

(5) in the passage step, the operation and dissociation of matrigel are not needed, and the digestion passage of the cells can be completed within 10-15 minutes;

(6) the culture cost is controllable: the primary lung cancer culture medium does not need to add expensive factors such as Wnt agonist, R-spondin family protein, BMP inhibitor and the like, so that the cost of cell culture is saved;

(7) compared with organoid technology, the technology does not need to embed cells in matrigel like organoid technology, and the technical operation steps are simple and easy to implement;

(9) the lung cancer epithelial cells obtained by the technical culture have large quantity and high homogenization degree, are suitable for screening new candidate compounds in high flux, and provide high-flux drug in-vitro sensitivity function test for patients.

With the cell culture medium of the present embodiment, lung cancer epithelial cells derived from a human or other mammal, including lung cancer tumor cells, normal lung cancer epithelial cells, lung cancer epithelial stem cells, or tissues containing at least any of these cells, can be cultured. Meanwhile, the culture medium of the technology can also be used for developing a kit for in vitro lung cancer primary cell amplification culture.

The cells obtained by the culture method of the present embodiment can be applied to regenerative medicine, research of basic medicine of lung cancer epithelial cells, screening of drug response, development of new drugs derived from lung cancer diseases, and the like.

Drawings

FIGS. 1A-1F are graphs showing the effect of different factors in culture on lung cancer primary cell proliferation.

FIG. 2 is a graph showing the effect of different factor increases in culture medium on lung cancer primary cell proliferation.

FIGS. 3A-3G are graphs showing the effect of concentration of each additive factor on proliferation of lung cancer primary cells.

FIGS. 4A and 4B are photographs of lung cancer tumor cells obtained by culturing cells isolated from 1 lung cancer clinical tissue specimen (No. 0B0003) with the medium LM of the present invention up to day 5 and day 10, respectively, under an inverted microscope.

FIG. 5 is a summary of the primary culture period and the number of cells obtained for 16 samples.

FIG. 6 is a graph showing the growth of cells isolated from 8 cases of lung cancer puncture specimens (Nos. 0B0002, 0B0004, 0B0006, 0B0009, 0B0010, 0B0011, 0B0012, and 0B0013) by continuous culture in LM medium.

FIG. 7 is a graph showing the total number of cells obtained by culturing cells isolated from 1 clinical tissue specimen (No. 0B0004) for lung cancer in 5 different culture media.

FIG. 8 is a graph showing a comparison of cell growth curves obtained by culturing cells isolated from 1 clinical tissue specimen (No. 0B0004) of lung cancer in 5 different culture media.

FIG. 9A is a photograph showing the staining of cell nuclei with a nonspecific fluorescent dye DAPI after cells isolated from 1 case of a lung cancer surgically excised specimen (No. 0B0015) were cultured with the medium LM of the present invention to obtain lung cancer tumor cells, FIG. 9B is a photograph showing the staining of lung cancer tumor cells obtained as described above with the lung adenocarcinoma-specific antibody NapsinA, and FIG. 9C is a photograph showing the combination of FIGS. 9A and 9B.

FIGS. 10A to 10D are graphs comparing immunohistochemical results of primary tissue cells obtained from 1 lung cancer surgically excised sample (No. 0B0004) and lung cancer tumor cells obtained by culturing the cells in the medium LM of the present invention.

Fig. 11 shows a dose-effect graph of lung cancer tumor cells cultured to the first and fifth generations using the medium LM of the present invention on different targeted drugs from cancer tissue samples (No. 0B0011) obtained from the puncture of the same lung cancer patient.

Detailed Description

In the present specification, the epithelial cells include differentiated epithelial cells and epithelial stem cells obtained from epithelial tissues. "epithelial stem cells" refer to cells having long-term self-renewal ability and differentiation into epithelial cells, and refer to stem cells derived from epithelial tissues. Examples of the epithelial tissue include 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, breast, salivary gland, lacrimal gland, prostate, hair root, trachea, and lung. Among them, the cell culture medium of the present embodiment is preferably a medium for lung cancer epithelial cells.

In the present specification, the term "epithelial tumor cell" refers to a cell obtained by tumorigenization of a cell derived from the above-mentioned epithelial tissue.

In the present specification, the term "organoid" refers to a three-dimensional solid tissue body similar to an organ, which is formed by spontaneously organizing and aggregating cells at a high density in a controlled space.

[ preparation example of MST1/2 kinase inhibitor ]

In the present specification, an inhibitor of MST1/2 kinase refers to any inhibitor that directly or indirectly down-regulates MST1/2 signaling. In general, inhibitors of MST1/2 kinase, for example, bind to and reduce the activity of MST1/2 kinase. Due to the structural similarity of MST1 and MST2, MST1/2 kinase inhibitors may also be compounds that bind to and reduce the activity of MST1 or MST2, for example.

Preparation of MST1/2 kinase inhibitor Compound 14- ((7- (2, 6-difluorophenyl) -5, 8-dimethyl-6-

oxo

5,6,7, 8-tetrahydropteridin-2-yl) amino) benzenesulfonamide 1

Methyl 2-amino-2- (2, 6-difluorophenyl) acetate (a 2): after 2-amino-2- (2, 6-difluorophenyl) acetic acid (2.0 g) was added to a round-bottom flask, methanol (30 ml) was added, followed by dropwise addition of thionyl chloride (1.2 ml) under ice-cooling. The reaction system was reacted at 85 ℃ overnight. After the reaction was complete, the solvent was evaporated to dryness under reduced pressure to give a white solid which was used directly in the next step.

Methyl 2- ((2-chloro-5-nitropyrimidin-4-yl) amino) -2- (2, 6-difluorophenyl) acetate (a 3): after addition of methyl 2-amino-2- (2, 6-difluorophenyl) acetate (2 g) to a round bottom flask was added acetone (30 ml) and potassium carbonate (2.2 g), the system was cooled to-10 ℃ with an ice salt bath, followed by slow addition of a solution of 2, 4-dichloro-5-nitropyrimidine (3.1 g) in acetone. The reaction was stirred at room temperature overnight. After the reaction, the reaction mixture was filtered, the solvent was removed from the filtrate under reduced pressure, and the residue was purified by pressure silica gel column chromatography to obtain Compound A3. LC/MS: m + H359.0.

2-chloro-7- (2, 6-difluorophenyl) -7, 8-dihydropteridin-6 (5H) -one (a 4): 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 was stirred at 60 ℃ for two hours. After the reaction was completed, the solvent was evaporated under reduced pressure, and the obtained product was neutralized to be alkaline with saturated sodium bicarbonate. The mixture was extracted with ethyl acetate, and the organic phase was washed with water and saturated brine, respectively, and then dried over anhydrous sodium sulfate. Filtering the organic phase, and evaporating to dryness under reduced pressure to obtain a crude product. Washing the crude product with diethyl ether to obtain compound A4. LC/MS: m + H297.0.

2-chloro-7- (2, 6-difluorophenyl) -5, 8-dimethyl-7, 8-dihydropteridin-6 (5H) -one (a 5): 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, cooled to-35 deg.C, methyl iodide (0.9 ml) was added followed by sodium hydride (615 mg) and the reaction was stirred for an additional two hours. After the reaction, water was added to quench, ethyl acetate was used for extraction, and the organic phase was washed with water and saturated brine, respectively, and then dried over anhydrous sodium sulfate. Filtering the organic phase, and evaporating to dryness under reduced pressure to obtain a crude product. Washing the crude product with diethyl ether to obtain compound A5. LC/MS: m + H325.0.

4- ((7- (2, 6-difluorophenyl) -5, 8-dimethyl-6-oxo-5, 6,7, 8-tetrahydropteridin-2-yl) amino) benzenesulfonamide (1): to a round-bottom flask were 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 ℃ overnight. After the reaction is finished, filtering, and washing by methanol and ether to obtain the compound 1. LC/MS: m + H461.1.

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

Other MST1/2 inhibitor compounds of the invention were synthesized in analogy to compound 1 and their structural and mass spectral data are shown in the table below.

[ example 1]

Isolation of human primary lung carcinoma epithelial cells

Lung cancer tissue samples were obtained from three described and consented patients with lung cancer tumors who had been surgically resected with cancer tissue samples numbered 0B0010, 0B0011, 0B0012, respectively. An example of the sample (No. 0B0010) will be described below.

The collection of the tissue sample is performed within half an hour after surgical resection or biopsy of the patient. More specifically, under sterile conditions, a tissue sample of a non-necrotic area is excised and its volume is 0.5cm³The resulting mixture was placed in a pre-chilled 4mL tissue transfer solution (see Table 1 for specific formulations) contained in a 5mL plastic sterile lidded cryopreservation tube (purchased from Jiety, Guangzhou), and transported to the laboratory in the cold chain (0-10 ℃).

TABLE 1 tissue transport fluid formulations

TABLE 2 tissue digestive juice formulation

Tissue digestive juice component	Suppliers of goods	Final concentration
			HBSS	Gibco
	50% (by volume)
		RPMI-1640	Corning	50% (by volume)
Collagenase II	Sigma				2mg/mL
		Collagenase IV	Sigma	2mg/mL
Deoxyribonucleic acid I	Sigma				50U/mL
		Hyaluronidase	Sigma	0.5mg/mL
Calcium chloride	Shanghai worker				0.33mg/mL
		Bovine serum albumin	Shanghai worker	10mg/mL

In the biosafety cabinet, the tissue specimen (No. 0B0010) was transferred to a 100mm cell culture dish (purchased from nist), rinsed with a tissue transfer solution, and the surface of the tissue specimen was washed with residual blood and removed of excess tissue such as fat. Transferring the tissue sample after being moistened into another new 100mm culture dish, adding 2mL of transport solution, and cutting the tissue sample into pieces with the volume less than 3mm by using a sterile surgical blade and surgical forceps³The tissue fragment of (a).

Transfer the tissue sample pieces to a 15mL centrifuge tube and centrifuge at 1500rpm for 4 minutes using a bench top centrifuge (Sigma Co., 3-18K); the supernatant was discarded, and the tissue transfusion solution and the tissue digestion solution (5 mL of tissue digestion solution per 10mg of tissue, see Table 2 for specific formulation) were added at a ratio of 1:1, the sample was labeled with the number, the membrane was sealed, and the sample was digested with a constant temperature shaker (Zhichu apparatus ZQLY-180N) at 37 ℃ and 300 rpm, and the completion of digestion was observed at 1 hour intervals.

After digestion was complete, undigested tissue pellet was filtered through a 70 μm filter, the tissue pellet on the filter was rinsed with tissue transfusion solution, the remaining cells were flushed into a centrifuge tube and centrifuged at 1500rpm for 4 minutes.

The supernatant was discarded, and the remaining cell pellet was observed to see whether or not it contained blood cells, and if any, 3mL of a blood cell lysate (purchased from Sigma) was added, mixed well, lysed at 4 ℃ for 15 minutes, shaken once for 5 minutes, taken out after lysis, and centrifuged at 1500rpm for 4 minutes. The supernatant was discarded to obtain primary lung cancer cells after digestion and isolation, and the cells were resuspended in Basal Medium (BM) prepared by adding 0.2 vol% Primocin (obtained from Invivogen at 50mg/mL) in DMEM/F-12 medium to obtain a final concentration of 100. mu.g/mL. The total number of cells was 10 ten thousand by counting using a flow cytometer (JIMBIO FIL, Ohio).

Two other lung cancer tumor tissue samples were isolated in the same manner as above, and the total number of cells obtained was 10 ten thousand (0B0011) and 8 ten thousand (0B0012), respectively.

[ example 2]

Optimization of primary lung cancer epithelial cell culture medium

(1) Effect of different factors

Cultured NIH-3T3 cells (purchased from ATCC, cultured using DMEM medium containing 10% fetal bovine serum) were digested with 0.25% pancreatin (purchased from Thermo Fisher), the digestion was terminated with DMEM medium containing 5% (v/v) fetal bovine serum (purchased from Eikey), 100U/mL penicillin and 100. mu.g/mL streptomycin (purchased from Corning), and collected in a 15mL centrifuge tube, and after centrifugation at 1500rpm for 4 minutes, the supernatant was discarded. The cell pellet after the centrifugation was resuspended in the above DMEM culture solution containing 10% fetal bovine serum, counted using a flow cytometer (JIMBIO FIL, Ohio, Jiangsu, Zong microbial technology Co., Ltd.), irradiated with gamma rays at an irradiation dose of 35Gy, and then irradiated at 2X 10⁴Per cm²Is inoculated into a culture vessel. Culturing in an incubator at 37 ℃ until the cells adhere to the wall. Prior to seeding the primary cells, the medium in the culture vessel was removed.

Basal medium (abbreviated BM): BM was prepared by adding 0.2 vol% Primocin (purchased from Invivogen at 50mg/mL) to a commercially available DMEM/F-12 medium to give a final concentration of 100. mu.g/mL.

Then, different kinds and concentrations of additive factors (table 3) were added to the Basal Medium (BM) to prepare lung cancer epithelial cell culture media containing different additive components.

TABLE 3 preparation of media of different composition (final concentration)

The culture medium with different components is added according to the volume of 500 mu l/porePre-plated 48-well culture plates with NIH-3T3 cells after gamma irradiation. Lung cancer tumor cells (No. 0B0014) isolated from lung cancer tissue by the same method as in example 1 were cultured at 4X 10⁴The number of cells per well was inoculated into the above 48-well culture plate preplaced with NIH-3T3 cells after gamma-ray irradiation, surface-sterilized and then placed at 37 ℃ with 5% CO₂Incubators (purchased from semer), cultured the same number of freshly isolated lung cancer tumor cells (No. 0B0014) under different media formulation conditions. The medium replacement and feeder cell supplementation were performed every 4 days after the start of the culture. After 7 days of culture, cell counts were performed. As experimental control, Basal Medium (BM) without any additives was used. The results are shown in FIGS. 1A-F.

The ordinate in the figure represents the ratio of the number of cells obtained after cultivation in the different media to the number of cells obtained after cultivation in the basal medium BM. As shown, the addition of different factors at different concentrations in Table 3 on the basis of BM produced different effects on cell proliferation. Wherein, under a specific concentration range, the B27 additive, the N2 additive, the insulin-transferrin-selenium compound, the hepatocyte growth factor, the insulin sample growth factor 1, the fibroblast growth factor 7, the neuregulin-1, the compound 1 and the Y27632 have certain promotion effects on cell proliferation.

(2) Influence of different factors in culture medium increasing on lung cancer primary cell proliferation obtained by the method

Different small molecules, additives and growth factors (shown in table 4) are sequentially added into the basal medium BM to prepare the lung cancer epithelial cell culture medium containing different additive components.

TABLE 4 preparation of the media of the different components (final concentration)

Different components of culture medium are added into 48-hole culture plates pre-paved with NIH-3T3 cells after gamma ray irradiation according to 500 mul/hole volume, and BM culture medium is used as experimental control. Will be provided withLung cancer tumor cells (No. 0B0016) isolated from lung cancer tissue according to the method of example 1 at 4X 10⁴The number of cells per well was inoculated into the above 48-well culture plate preplaced with NIH-3T3 cells after gamma-ray irradiation, surface-sterilized and then placed at 37 ℃ with 5% CO₂Incubators (purchased from semer), cultured the same number of freshly isolated lung cancer tumor cells (No. 0B0016) under different media formulation conditions. After 7 days of culture, cell counts were performed. The experimental results are shown in fig. 2.

As shown in the figure, NO.7 was determined to be the most preferred medium for the culture of expanded lung cancer primary cells (hereinafter abbreviated as LM).

(3) Proliferation of primary cells of lung cancer obtained in this patent by different concentrations of added factors

Lung cancer epithelial cells derived from a cancer tissue were isolated from a cancer tissue (sample No. 0B0002) of a lung cancer patient by the same method as in example 1. Subsequently, the lung cancer tumor cells derived from the cancer tissue were counted by a flow cytometer (jiamboi FIL, jiangsu microbial technologies, ltd.) to obtain the total number of cells. Then press 4X 10⁴Per cm²The cells were densely seeded in 12-well plates pre-plated with NIH-3T3 cells after gamma irradiation. Adding 2mL of the prepared primary lung cancer epithelial cell culture medium NO.7(LM) into a 12-well plate, placing at 37 deg.C and 5% CO₂Incubators (purchased from Saimeri fly) were used for culturing. When the cells grown in the culture plate reached about 80% of the basal area, the culture medium supernatant in the 12-well plate was discarded, 0.5mL of 0.25% pancreatin (purchased from Thermo Fisher) was added for digestion for 1 minute, then 0.25% pancreatin was aspirated, 0.5mL of 0.05% pancreatin was added for cell digestion, and the mixture was incubated at room temperature for 5 to 20 minutes until completion of cell digestion was observed under a microscope (Invitrogen EVOS M500), i.e., 1mL of a DMEM/F12 culture solution containing 5% (v/v) fetal bovine serum, 100U/mL penicillin and 100. mu.g/mL streptomycin was used to stop digestion and collected in a 15mL centrifuge tube, and after centrifugation at 1500rpm for 4 minutes, the supernatant was discarded. Resuspending the centrifuged cell pellet in a basal medium BM, and counting the cell pellet with a flow cytometer (JIMBIO FIL, Ohio, Jiangsu, Ohio, Ltd.) to obtain cellsAnd (4) total number. The cells obtained were used in the following culture experiments.

Next, the following 7 formulations of media were prepared for the experiments:

formula 1: medium LM contains no compound 1;

and (2) formula: medium LM contains no Y27632;

and (3) formula: medium LM contains no insulin-like growth factor 1;

and (4) formula: the LM component of the culture medium does not contain hepatocyte growth factor;

and (5) formula: the LM component of the culture medium does not contain insulin-transferrin-selenium compound;

and (6) formula: the LM component of the culture medium does not contain epidermal growth factors;

and (3) formula 7: the medium LM component does not contain neuregulin-1.

The digested cell suspensions were diluted with the formulations 1-7, respectively, and seeded into 48-well plates pre-plated with NIH-3T3 cells irradiated with gamma rays in a volume of 250 μ l per well of 1 ten thousand cells.

When the culture medium of formula 1 is used, 250 microliters of the prepared compound 1 per well is added to 48-well plates inoculated with primary cells, respectively, and the final concentrations of the compound 1 are 40 μ M, 20 μ M, 10 μ M, 5 μ M, 2.5 μ M, 1.25 μ M, and 0.625 μ M, respectively; and control wells (BC) were set using medium of formula 1.

When the culture medium of formula 2 is used, 250 microliters of prepared Y27632 per well are added to 48-well plates inoculated with primary cells respectively, and the final concentrations of Y27632 are 40. mu.M, 20. mu.M, 10. mu.M, 5. mu.M, 2.5. mu.M, 1.25. mu.M and 0.625. mu.M respectively; and control wells (BC) were set using medium of formula 2.

When the culture medium of formula 3 is used, 250 microliters of prepared insulin-like growth factor 1 is respectively added into 48-well plates inoculated with primary cells, and the final concentrations of the insulin-like growth factor 1 are respectively 80ng/ml, 40ng/ml, 20ng/ml, 10ng/ml, 5ng/ml, 2.5ng/ml and 1.25 ng/ml; and control wells (BC) were set using medium of formula 3.

When the culture medium of formula 4 is used, 250 microliters of prepared hepatocyte growth factor is respectively added into a 48-well plate inoculated with primary cells, and the final concentrations of the hepatocyte growth factor are respectively 80ng/ml, 40ng/ml, 20ng/ml, 10ng/ml, 5ng/ml, 2.5ng/ml and 1.25 ng/ml; and control wells (BC) were set using medium of formula 4.

When the culture medium of the formula 5 is used, 250 microliters of prepared insulin-transferrin-selenium compound is respectively added into 48-well plates inoculated with primary cells, and the final concentrations of the insulin-transferrin-selenium compound are respectively 1:1600, 1:800, 1:400, 1:200, 1:100, 1:50 and 1: 25; and control wells (BC) were set using medium of formula 5.

When the culture medium of formula 6 is used, 250 microliters of prepared epidermal growth factor per well is respectively added into a 48-well plate inoculated with primary cells, and the final concentrations of the epidermal growth factor are respectively 80ng/ml, 40ng/ml, 20ng/ml, 10ng/ml, 5ng/ml, 2.5ng/ml and 1.25 ng/ml; and control wells (BC) were set using medium of formula 6.

When the culture medium of the formula 7 is used, 250 microliters of prepared neuregulin-1 is respectively added into a 48-well plate inoculated with primary cells, and the final concentrations of the neuregulin-1 are respectively 80ng/ml, 40ng/ml, 20ng/ml, 10ng/ml, 5ng/ml, 2.5ng/ml and 1.25 ng/ml; and control wells (BC) were set using medium of formula 7.

When the cells were expanded to about 85% of the 48 wells and digested to count, the ratio was calculated with reference to the number of cells in the control well (BC), and the results are shown in FIGS. 3A to 3G, respectively. In FIGS. 3A to 3G, the ratio is the ratio of the number of cells obtained by one-pass culture using each medium to the number of cells obtained by one-pass culture using the corresponding control well. The ratio is more than 1, which indicates that the proliferation promoting effect of the prepared culture medium containing factors or small molecular compounds with different concentrations is better than that of a control hole culture medium; if the ratio is less than 1, the proliferation promoting effect of the prepared culture medium containing factors or small molecular compounds with different concentrations is weaker than that of the culture medium of the control hole.

According to the results of FIGS. 3A to 3G, the content of Compound 1 is preferably 0.625. mu.M to 20. mu.M, more preferably 0.625. mu.M to 10. mu.M; the content of Y27632 is preferably 1.25-20. mu.M, more preferably 2.5-10. mu.M; the content of the insulin-like growth factor 1 is preferably 1.25ng/ml to 80ng/ml, and more preferably 5ng/ml to 80 ng/ml; the content of the hepatocyte growth factor is preferably 5ng/ml to 80ng/ml, and more preferably 20ng/ml to 80 ng/ml; the volume concentration of the insulin-transferrin-selenium compound is preferably 1: 25-1: 200 (the content of each insulin/transferrin/sodium selenite is 2.5-20 mug/ml-1.25-10 ng/ml), and more preferably 1: 25-1: 50 (the content of each insulin/transferrin/sodium selenite is 10-20 mug/ml-5-10 ng/ml); the content of the epidermal growth factor is preferably 10 ng/ml-80 ng/ml, and more preferably 10 ng/ml-40 ng/ml; the content of the neuregulin-1 in the culture medium is preferably 10-80 ng/ml, and more preferably 20-80 ng/ml.

[ example 3]

Culture of primary lung cancer tumor cells derived from human lung cancer tissue

Lung cancer epithelial cells derived from a cancer tissue were isolated from a cancer tissue (sample No. 0B0003) of a lung cancer patient by the same method as in example 1. Subsequently, the lung cancer tumor cells derived from the cancer tissue were counted by a flow cytometer (jiamboi FIL, jiangsu microbial technologies, ltd.) to obtain the total number of cells. Then press 4X 10⁴Per cm²The cells were densely seeded in 12-well plates pre-plated with NIH-3T3 cells after gamma irradiation. Adding 2mL of prepared primary lung cancer epithelial cell culture medium LM into 12-well plate, placing at 37 deg.C and 5% CO₂Incubators (purchased from Saimeri fly) were used for culturing.

FIG. 4A shows a schematic view of a 4X 10 display device according to the present embodiment⁴Per cm²12-well plates pre-plated with NIH-3T3 cells after gamma irradiation were density-inoculated, and an under-lens photograph (100-fold inverted phase contrast microscope photograph) was taken from the culture to day 5 after the start of inoculation. Under the observation of a microscope, the cultured cancer tissue-derived primary lung cancer tumor cells form larger clones. FIG. 4B is an under-the-lens photograph (photographed by a 100-fold inverted phase contrast microscope) of the present example from the 10 th day of culture after inoculation, and the cells were overgrown in the visual field. As can be seen from the two graphs of FIGS. 4A and 4B, the primary cells of lung cancer obtained after isolation were cultured in vitro for 5 days and under the microscope, significant colony formation was observed, andafter 10 days of amplification, the cell number is obviously amplified, which indicates that the technology of the invention is a high-efficiency technology for in vitro amplification of lung cancer epithelial cells.

[ example 4]

Primary culture period and cell count statistics of lung cancer and calculation of Probability Doubling (PD) value

Lung cancer primary cells were obtained by digesting lung cancer punctured tissue samples (sample Nos. 0B 0001-0B 0016) by the method of example 1. For the obtained lung cancer primary cells, LM medium was used according to a viable cell density of 4X 10⁴Per cm²The cells were seeded in a 12-well plate and cultured, digested and counted after the cells were expanded to 85%, and the number of days of culture until digestion was recorded as one culture cycle. As shown in FIG. 5, the average number of cells at the start of culture of 16 lung cancer samples was 7.2 ten thousand, the average number of cells obtained after the first amplification was 121.2 ten thousand, and the required average culture period was 12.6 days.

Under the experimental conditions, 8 samples of sample numbers 0B0002, 0B0004, 0B0006, 0B0009, 0B0010, 0B0011, 0B0012 and 0B0013 were continuously cultured, the cells obtained by amplification were subjected to different generations of amplification, digestion was performed for each generation, the corresponding culture period was counted, PD was calculated according to the formula Poultion Doubling (PD) ═ 3.32 log10 (total number of cells after digestion/initial number of seeded cells), as shown in fig. 6, the abscissa represents the number of days of cell culture, the ordinate represents the cumulative cell growth multiple, the fold of cells in the culture period is represented, and the larger the number represents the number of times that cells are amplified in a certain period, that is, the larger the number of cells obtained by amplification, and the slope represents the rate of cell amplification.

As can be seen from FIG. 6, when the lung cancer primary cell culture medium of the present invention was used to culture the above 8 samples, the cell expansion rate after 58 days of expansion remained substantially unchanged, and the capacity of continuous expansion was still maintained.

[ example 5]

Proliferation promoting effect of different culture media on primary lung cancer tumor cells derived from lung cancer tissues

(1) Comparison of the proliferation Effect of Primary cells on different media

Primary lung cancer epithelial cell culture medium LM and basal medium BM as a control were prepared in the same manner as in example 2. In addition, as another control example, medium FM used in the technical literature of cell condition reprogramming was prepared, and the formulation steps are shown in (Liu et al, nat. protocol., 12(2): 439. cndot. 451, 2017), and the medium formula is shown in Table 5. Meanwhile, as an additional control example, a lung cancer primary cell culture medium LM1 was prepared, formulated to replace insulin-transferrin-selenium complex with B27 supplement at a 1:50 volume ratio on the LM basis. In addition, as another control example, a commercial culture medium, EpiCult, was purchased from STEMCELL^TMPlus Medium, also referred to as "Epi Medium" below), the Medium formulation is shown in Table 6.

TABLE 5 Medium (FM) Components used in the technical literature for the conditional reprogramming of cells

TABLE 6 commercial culture Medium EpiCult^TMPlus Medium (Epi) component

Media composition	Suppliers of goods	Final concentration
			EpiCult^TM Plus Basal Medium	STEMCELL	99Volume%
EpiCult^TM Plus Supplement	STEMCELL					1% by volume

Primary lung cancer tumor cells derived from lung cancer tissue (No. 0B0006) were obtained in the same manner as in example 1. Then, the same density (4X 10) was used⁴Per cm²) The culture was carried out under the following 5 culture conditions, respectively:

A. the technology of the invention is as follows: by 4X 10⁴Per cm²Inoculating density, inoculating primary lung cancer tumor cells into a 24-well plate pre-paved with NIH-3T3 cells (purchased from ATCC company) after gamma-ray irradiation, and culturing by adopting 1mL of the primary lung cancer epithelial cell culture medium LM of the invention;

B. cell condition reprogramming techniques: by 4X 10⁴Per cm²Inoculation density primary lung cancer tumor cells were inoculated onto NIH-3T3 cells (purchased from ATCC) pre-plated after gamma irradiation, and cultured in 24-well plates using 1mL of cell condition reprogramming technical medium FM (see (Liu et al, nat. Protoc., 12(2): 439. plus 451, 2017);

C. by 4X 10⁴Per cm²Inoculating density primary lung cancer tumor cells into a 24-well plate pre-paved with NIH-3T3 cells (purchased from ATCC company) after gamma-ray irradiation, and culturing in the 24-well plate by adopting 1mL of a culture medium LM 1;

D. by 4X 10⁴Per cm²Primary lung cancer tumor cells were seeded into 24-well plates and cultured in 24-well plates using 1mL of a commercial culture medium, Epi.

E. By 4X 10⁴Per cm²Primary lung cancer tumor cells were inoculated into 24-well plates pre-plated with gamma-irradiated NIH-3T3 cells (purchased from ATCC) and cultured in 24-well plates using 1mL of basal medium BM.

In the above five cultures, the cells cultured under the five culture conditions were changed every 5 days. The state of cell formation cloning and cell proliferation was observed in 24-well plates under each medium culture, and the growth of the cells was recorded by photographing using a microscope (EVOS M500, Invitrogen).

For primary lung cancer tumor cells cultured by the technique of the present invention (No. 0B0006), when the cell growth in the culture plate reached about 80% of the basal area, the culture medium supernatant in the 24-well plate was discarded, 0.5mL of 0.25% pancreatin (purchased from Thermo Fisher) was added and digested for 1 minute, then 0.25% pancreatin was aspirated, 0.5mL of 0.05% pancreatin was added and cell digestion was performed, and the cell was incubated at 37 ℃ for 10 minutes until completion of cell digestion was observed under a microscope (EVOS M500 from Invitrogen), i.e., 1mL of a DMEM/F12 culture solution containing 5% (v/v) fetal bovine serum, 100U/mL of penicillin and 100. mu.g/mL of streptomycin was used to terminate the digestion and collected in a 15mL centrifuge tube, and the supernatant was discarded after centrifugation at 1500rpm for 4 minutes. The cell pellet after centrifugation was resuspended in the culture medium of the present invention, and the total number of cells was 44.6 ten thousand by counting with a flow cytometer (JIMBIO FIL, Jiangsu Zong microbial technology Co., Ltd.). The cells cultured under the other 4 culture conditions were digested and counted in the same manner as described above, and the total number of cells cultured using the media FM, LM1, Epi and BM was 29.6 ten thousand, 39.6 ten thousand, 25 ten thousand and 6.9 ten thousand, respectively.

FIG. 7 is a plot of the total number of cells expanded under different conditions for cells numbered 0B 0006.

(2) Continuous culture of primary lung cancer tumor cells by different culture media and drawing of growth curve

Primary lung cancer epithelial cell culture medium LM, and culture media FM, LM1, Epi, and BM as controls were obtained using the same method as in example (1).

Primary lung cancer tumor cells (No. 0B0004) derived from lung cancer tissue were cultured in five media, respectively, and subjected to digestion passaging and counting, using the same method as in example (1).

When the cells after passage grow again to about 80% of the plate bottom area in the culture plate, the procedure is again as described aboveMethod cells obtained from the culture were collected by digestion and counted. Similarly by 4X 10⁴The cells were inoculated at a density per well and cultured continuously.

The following formula is the calculation formula of the amplification factor (amplification Doubling) of primary lung cancer epithelial cells under different technical culture conditions:

Population Doubling(PD)＝3.32*log₁₀(total number of cells after digestion/initial number of seeded cells) see the formula (Chapman et al, Stem Cell Research)&Therapy 2014,5:60)。

FIG. 8 shows the growth curves of cell 0B0004 under five different culture conditions using Graphpad Prism software. The abscissa represents the number of days in which the cells are cultured, and the ordinate represents the cumulative cell proliferation fold, which represents the fold of the cells amplified during the culture period, wherein a larger value represents a larger number of times the cells are amplified within a certain period, i.e., a larger number of cells are amplified, and the slope represents the rate of cell amplification. From the figure, it can be confirmed that the proliferation rate of lung cancer epithelial cells cultured by the medium LM and LM1 of the present invention is superior to that of the other three culture conditions, and it can be confirmed that the present invention can continuously culture primary lung cancer epithelial cells.

[ example 6]

Identification of cancer tissue-derived primary lung cancer tumor cells

(1) Immunofluorescence identification of primary lung cancer tissue and lung cancer cells after subculture

Lung cancer epithelial cells derived from a cancer tissue were isolated from a cancer tissue (sample No. 0B0015) of a lung cancer patient by the same method as in example 1. Subsequently, the lung cancer tumor cells derived from the cancer tissue were counted by a flow cytometer (jiamboi FIL, jiangsu microbial technologies, ltd.) to obtain the total number of cells. Then press 4X 10⁴Per cm²The cells were densely seeded in 24-well plates pre-plated with NIH-3T3 cells after gamma irradiation, while circular cell fragments for immunofluorescent staining (purchased from Saimer fly) were previously placed in the 24-well plates. Adding 1mL of prepared primary lung cancer epithelial cell culture medium LM into 24-well plate, placing at 37 deg.C and 5% CO₂Cultivation in incubator (purchased from Saimeri fly)And (5) nourishing.

When 80% of the basal area of the cells in the 24-well plate was expanded, the culture solution was discarded, and the cells were fixed on 4% formaldehyde ice for 30 minutes. PBS (from shanghai bio-workers) for 5 min x 3 times. Discard PBS, add the permeation solution, avoid light, break the membrane for 30 minutes by shaking table (about 100 rpm), wash 5 minutes x 3 times with PBS. A5% strength by volume BSA (ex Shanghai) solution was then prepared for blocking using PBS + 0.3% Triton X-100 (ex Shanghai) and blocked for 30 minutes at 37 ℃.

PBS + 0.3% Triton X-100 was prepared in advance for antibody dilution, the lung adenocarcinoma specific antibody NapsinA (purchased from CST) was diluted at a ratio of 1:50, the blocking solution was discarded, the prepared primary antibody dilution was added, and the mixture was incubated overnight in a refrigerator at 4 ℃. The cells were removed at 4 ℃ and allowed to equilibrate to room temperature, incubated for an additional 1 hour at 37 ℃ and washed 5 minutes x 3 times with PBS.

PBS + 0.3% Triton X-100 was prepared in advance for dilution of the secondary antibody, a murine fluorescent secondary antibody (purchased from Saimer fly) with an excitation light of 488nm was diluted at a ratio of 1:1000, incubated for 1 hour at room temperature in the dark, and washed with PBS for 5 minutes X3 times.

The nonspecific fluorescent dye DAPI (purchased from Sigma) was diluted with PBS at a volume ratio of 1:1000, stained for 5 minutes at room temperature in the dark, and washed 5 minutes with PBS x 3 times. Images were taken under a microscope (EVOS M500, Invitrogen) and recorded by photography.

FIGS. 9A-C are pictures taken under different conditions with a 10-fold objective lens, wherein FIG. 9A is a picture of the nuclei stained with the nonspecific fluorescent dye DAPI, and FIG. 9B is a picture of the nuclei stained with the lung adenocarcinoma-specific antibody NapsinA (localized to the cytoplasm). Fig. 9C is a picture obtained by combining fig. 9A and 9B. As shown, the location of the marker nuclei in FIG. 9A and the cytoplasm in FIG. 9B were labeled with specific antibodies, indicating that the cultured cells were lung adenocarcinoma cells, consistent with the clinical pathological diagnosis.

(2) Immunohistochemical identification of primary lung cancer tissues and lung cancer cells after subculture

Cancer tissue (sample No. 0B0004) having a size of about mung bean grain was taken from a clinical surgical resection specimen of one lung cancer patient, and fixed by immersing in 1mL of 4% paraformaldehyde. The lung cancer epithelial cells (sample No. 0B0004) were obtained from the remaining cancer tissues by the same method as in example 1. The sample 0B0004 was continuously cultured up to passage 5 using the medium LM of the present invention using the method of example 3.

The expression of important biomarkers related to lung cancer in the sample 0B0004 original tissue and primary cells obtained by continuous culture to the 3 rd generation is detected by an immunohistochemistry method. The 4% paraformaldehyde fixed tissue was embedded in paraffin and sliced into 4 μm thick tissue sections with a microtome. Conventional immunohistochemical detection was then performed (see, for specific steps, Li et al, Nature communication, (2018)9: 2983). The primary antibodies used were thyroid transcription factor 1(TTF-1) (from CST) and ki-67 antibody (from R & D).

FIGS. 10A to 10D are graphs comparing immunohistochemical results of primitive tissue cells and lung cancer tumor cells obtained by culturing the cells in the medium LM of the present invention. FIGS. 10A and 10B are pictures of a lung cancer tissue and a cell marker thyroid transcription factor 1 antibody obtained after the culture for amplification, respectively, and FIGS. 10C and 10D are pictures of a lung cancer tissue and a cell marker ki-67 antibody obtained after the culture for amplification, respectively. From this, it was confirmed that when the lung cancer tumor cells (sample No. 0B0004) cultured by the technique of the present invention were cultured up to the 5 th generation, the expression of the biomarker related to lung cancer on the cells substantially matched the expression of the marker in the original tissue section derived from the cells. It is demonstrated that the cells cultured by the technology of the invention maintain the original pathological characteristics of the cancer tissues of the lung cancer patients.

[ example 7]

Xenograft tumorigenesis experiment of cancer tissue-derived primary lung cancer tumor cells in mice

Lung cancer tumor cells (No. 0B0003) were isolated from a cancer tissue of a patient pathologically diagnosed with lung cancer by the same method as in example 1, and 0B0003 was cultured by the method of example 3 using the medium LM of the present invention until the number of lung cancer tumor cells reached 1X 10⁷At one time, lung cancer tumor cells were digested and collected as described in example 5. The lung cancer tumor cell culture media LM and the preparation method thereof

(purchased from BD Biotech Co.) at a ratio of 1:1, and pipetting 100. mu.L of the medium mixed with Matrigel to 5X 10⁶Each lung cancer tumor cell was resuspended, injected into the lung cancer fat pad and right anterior axillary region of a 6-week-old female hyperimmune deficient mouse (NCG) mouse (purchased from Nanjing model animal institute), and the tumor formation volume and growth rate of the lung cancer tumor cells in the mouse were observed every three days.

On day 15 after tumor cell inoculation, tumor bodies are formed at two tumor cell inoculation positions of the mice, and the tumor proliferation in the mice is obvious from day 15 to day 36. This indicates that the lung cancer tumor cells derived from cancer tissue cultured by the culture method of the present invention have tumorigenicity in mice.

[ example 8]

Drug sensitivity function test of cancer tissue-derived lung cancer tumor cells

The following description will take a puncture sample of a lung cancer patient as an example, and will explain that lung cancer tumor cells cultured from a lung cancer tumor sample derived from a patient can be used for detecting the sensitivity of the lung cancer tumor cells of the patient to different drugs.

Primary lung cancer tumor cell plating: the isolated lung cancer tumor cell (No. 0B0011) cell suspension obtained by the method of example 1 was prepared at 4X 10⁴Per cm²The cells were densely seeded in 6-well plates pre-plated with NIH-3T3 cells after gamma irradiation. Adding 2mL of prepared primary lung cancer epithelial cell culture medium LM into 6-well plate, placing at 37 deg.C and 5% CO₂Incubators (purchased from Saimeri fly) were used for culturing. When the cells grown in the plates reached about 80% of the basal area, the culture supernatant in the 6-well plate was discarded, 1mL of 0.25% pancreatin (purchased from Thermo Fisher) was added for 1 minute for digestion, then 0.25% pancreatin was aspirated, 1mL of 0.05% pancreatin was added for cell digestion, and the plates were incubated at 37 ℃ for 10 minutes until complete digestion of the cells was observed under a microscope (EVOS M500 from Invitrogen), i.e., digestion was terminated with 2mL of DMEM/F12 medium containing 5% (v/v) fetal bovine serum, 100U/mL penicillin, and 100. mu.g/mL streptomycin, and collected to 15mL of supernatant removedAfter centrifugation at 1500rpm for 4 minutes in the vessel, the supernatant was discarded. The centrifuged cell pellet was resuspended in LM medium and counted using a flow cytometer (JIMBIO FIL, Ohio), to obtain a total number of 160 ten thousand cells in the first generation. Inoculating the cells in a 384-well plate according to the density of 2000-4000 cells/well, and allowing the cells to adhere to the wall overnight. The remaining cells continued to be at 4X 10⁴Per cm²Inoculating the cells into a 6-well plate pre-paved with NIH-3T3 cells irradiated by gamma rays for continuous passage for 4 times, digesting and terminating digestion according to the method, and counting the cells by using a flow image counter (JIMBIO FIL of Jiangsu Zongmi Microbiol Co., Ltd.) after resuspending the cells to obtain the total number of the cells of the fifth generation of 180 ten thousand. Inoculating the cells in a 384-well plate according to the density of 2000-4000 cells/well, and allowing the cells to adhere to the wall overnight.

II, drug gradient experiment:

(1) preparing a drug storage plate by adopting a concentration gradient dilution method: the drug was diluted 1:3 by pipetting 40. mu.L of each 10. mu.M of the mother solution of the drug to be tested as the highest concentration, pipetting 10. mu.L of the mother solution of the drug to be tested, adding the pipetted mother solution to a 0.5mL EP tube containing 20. mu.L of DMSO, and pipetting 10. mu.L of the pipette from the above EP tube to a second 0.5mL EP tube containing 20. mu.L of DMSO. Repeating the above method, sequentially diluting to obtain 7 concentrations required by dosing. Drugs at different concentrations were added to 384-well drug storage plates. Solvent control an equal volume of DMSO was added to each well as a control. In this example, the drugs to be tested were afatinib (available from MCE), gefitinib (available from MCE), oxitinib (available from MCE), and crizotinib (available from MCE). (2) Different concentrations of drug and solvent controls in 384-well drug storage plates were added to 384-well cell culture plates plated with lung cancer tumor cells using a high throughput automated workstation (Perkin Elmer JANUS), each with 3 replicates of the drug and solvent controls. The volume of drug added per well was 100 nL.

(3) And (3) detecting the activity of the cells: after 72 hours of administration, the chemiluminescence values of the cells after the drug addition culture were measured with a Cell Titer-Glo detection reagent (available from Promega), the chemiluminescence values reflect the Cell viability and the effect of the drug on the Cell viability, 10 μ L of the prepared Cell Titer-Glo detection solution was added to each well, and the chemiluminescence values were measured with a microplate reader (Envision, Perkin Elmer).

(4) And (3) detecting the activity of the cells: calculating the cell survival rate after different drug action cells according to the formula of cell survival rate (%). adding hole chemiluminescence value/control hole chemiluminescence value 100%, using Graphpad Prism software to map and calculating half inhibition rate IC₅₀And meanwhile, calculating the cell survival rate of different medicines corresponding to the maximum blood concentration Cmax in the human body.

(5) The results of the drug susceptibility testing are shown in figure 11.

Figure 11 shows the sensitivity of the 1 st and 5 th generation lung cancer tumor cells cultured from a surgically excised cancer tissue sample (No. 0B0011) from the same lung cancer patient to the targeted drugs afatinib, gefitinib, axitinib and crizotinib. The concentration of the four drugs in the human body is the maximum blood concentration Cmax on the abscissa of the graph corresponding to the marked line. The results show that the sensitivity of the cells of the same patient to different drugs at the maximum blood concentration in the human body is different, and the sensitivity of the cells of different generations to the same drug is basically consistent. According to the results, the effectiveness of the medicine in clinical use of lung cancer patients can be judged, and meanwhile, the sensitivity of tumor cells with different generations obtained by the culture method of the patent to the medicine can be proved to be stable.

Industrial applicability

The invention provides a culture medium and a culture method for culturing or amplifying primary lung cancer epithelial cells in vitro, and the cells obtained by culture can be applied to the curative effect evaluation and screening of medicaments. Thus, the present invention is suitable for industrial applications.

Although the present invention has been described in detail herein, the present invention is not limited thereto, and modifications can be made by those skilled in the art based on the principle of the present invention, and thus, it is to be understood that various modifications made in accordance with the principle of the present invention are within the scope of the present invention.

Claims

1. A primary cell culture medium for culturing lung cancer epithelial cells, comprising:

contains MST1/2 kinase inhibitor; insulin-like growth factor 1; an epidermal growth factor; hepatocyte growth factor; neuregulin-1; an additive selected from at least one of insulin-transferrin-selenium complex, B27 additive, and N2 additive; a ROCK kinase inhibitor selected from at least one of Y27632, fasudil, and H-1152,

the MST1/2 kinase inhibitor includes a compound of formula (I) or a pharmaceutically acceptable salt, or solvate thereof,

wherein the content of the first and second substances,

R₁selected from C1-C6 alkyl, C3-C6 cycloalkyl, C4-C8 cycloalkylalkyl, C2-C6 spirocycloalkyl, and optionally substituted with 1-2 independent R₆Substituted aryl, aryl C1-C6 alkyl and heteroaryl;

R₂and R₃Each independently selected from C1-C6 alkyl;

R₄and R₅Each independently selected from hydrogen, C1-C6 alkyl, C3-C6 cycloalkyl, C4-C8 cycloalkylalkyl, C1-C6 alkylhydroxy, C1-C6 haloalkyl, C1-C6 alkylamino C1-C6 alkyl, C1-C6 alkoxy C1-C6 alkyl, and C3-C6 heterocyclyl C1-C6 alkyl;

R₆selected from the group consisting of halogen, C1-C6 alkyl, C1-C6 alkoxy, and C1-C6 haloalkyl.

2. The primary cell culture medium of claim 1, wherein

R₁Selected from C1-C6 alkyl, C3-C6 cycloalkyl, C4-C8 cycloalkylalkyl, C2-C6 spirocycloalkyl, and optionally substituted with 1-2 independent R₆Substituted phenyl, naphthyl, benzyl and thienyl;

R₂and R₃Each independently selected from C1-C3 alkyl;

R₄and R₅Each independentlySelected from the group consisting of hydrogen, C1-C6 alkyl, C3-C6 cycloalkyl, C4-C8 cycloalkylalkyl, C1-C6 alkylhydroxy, C1-C6 haloalkyl, C1-C6 alkylamino C1-C6 alkyl, C1-C6 alkoxy C1-C6 alkyl, piperidinyl C1-C6 alkyl, and tetrahydropyranyl C1-C6 alkyl;

3. The primary cell culture medium of claim 1, wherein the MST1/2 kinase inhibitor comprises a compound of formula (Ia) or a pharmaceutically acceptable salt, or solvate thereof,

wherein the content of the first and second substances,

R₁selected from C1-C6 alkyl, optionally substituted with 1-2 independent R₆Substituted phenyl, optionally substituted with 1-2 independent R₆Substituted thienyl, and optionally substituted with 1-2 independent R₆A substituted benzyl group;

R₅selected from hydrogen, C1-C6 alkyl, and C3-C6 cycloalkyl;

R₆each independently selected from halogen, C1-C6 alkyl, and C1-C6 haloalkyl.

4. The primary cell culture medium of claim 3, wherein

R₁Is optionally substituted by 1-2 independent R₆Substituted phenyl;

R₅is hydrogen;

R₆preferably fluorine, methyl or trifluoromethyl.

5. The primary cell culture medium of claim 1, wherein the MST1/2 kinase inhibitor is selected from at least one of the following compounds, or a pharmaceutically acceptable salt thereof:

6. the primary cell culture medium of any one of claims 1 to 5, wherein:

the content of the MST1/2 kinase inhibitor is 0.625-20 mu M, preferably 0.625-10 mu M.

7. The primary cell culture medium of any one of claims 1-5, wherein:

the content of the insulin-like growth factor 1 is 1.25-80 ng/ml, and more preferably 5-80 ng/ml;

the content of the epidermal growth factor is 10-80 ng/ml, and more preferably 10-40 ng/ml;

the hepatocyte growth factor is 5-80 ng/ml, and more preferably 20-80 ng/ml;

the content of the neuregulin-1 is 10-80 ng/ml, and more preferably 20-80 ng/ml;

the content of the additive is preferably 1: 200-1: 25 by volume, and more preferably 1: 50-1: 25 by volume;

the content of the ROCK kinase inhibitor is preferably 1.25-20 mu M, and more preferably 2.5-10 mu M.

8. The primary cell culture medium of claim 7, wherein:

the additive is an insulin-transferrin-selenium compound, wherein the respective contents of insulin/transferrin/sodium selenite are preferably 2.5-20 mug/ml-1.25-10 ng/ml respectively, and more preferably 10-20 mug/ml-5-10 ng/ml respectively.

9. The primary cell culture medium of claim 7, wherein:

the ROCK kinase inhibitor is Y27632.

10. The primary cell culture medium of any one of claims 1 to 5, wherein:

is free of serum, bovine pituitary extract, Wnt agonist, R-spondin family protein, BMP inhibitor, nicotinamide and N-acetylcysteine.

11. The primary cell culture medium of any one of claims 1 to 5, wherein:

the lung cancer epithelial cells are selected from lung cancer tumor cells, normal lung cancer epithelial cells, and lung cancer epithelial stem cells.

12. A method for culturing lung cancer epithelial cells is characterized by comprising the following steps:

(1) preparing a primary cell culture medium according to any one of claims 1 to 11;

(2) pre-laying a culture vessel with the trophoblasts irradiated by X rays or gamma rays;

(3) inoculating a culture vessel pre-paved with trophoblasts to separate primary lung cancer epithelial cells from lung cancer tissues, and culturing by using the primary cell culture medium in the step (1).

13. A method of evaluating or screening a drug for treating a lung cancer disease, comprising the steps of:

(1) culturing according to the culture method of claim 12 to obtain lung cancer epithelial cells;

(2) selecting the medicine to be detected, and diluting the medicine to different medicine concentration gradients;

(3) and (2) adding the medicine after gradient dilution into the lung cancer epithelial cells cultured in the step (1), and detecting the cell viability.