CN115678849A - Culture medium for oral cancer organoid culture, and culture method and application thereof - Google Patents

Abstract

The invention relates to a culture medium for oral cancer organoid culture, comprising an MST1/2 kinase inhibitor, at least one cell culture additive selected from N2 and B27, a fibroblast growth factor, CHIR99021, an epidermal growth factor, insulin, an ITS cell culture additive, SB202190, Y27632, dexamethasone, glutaMAX, and non-essential amino acids. The invention also relates to a culture method of the oral cancer organoid and application thereof. By using the oral cancer organoid culture medium, the effective and rapid amplification of the oral cancer organoids can be realized, so that the amplified organoids keep the pathological characteristics of patients, the culture success rate and the amplification rate of the oral cancer organoids are improved, and a research basis can be provided for the personalized treatment of the patients.

Description

Culture medium for oral cancer organoid culture, and culture method and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a culture medium for culturing oral cancer organoids, a method for culturing oral cancer organoids by using the culture medium, and application of the culture medium in evaluating and screening curative effects of medicines.

Background

Oral cancer is a general term for malignant tumors occurring in the oral cavity. The oral cancer comprises cancers which are originally generated in oral cavity tongue (2/3 of the front of the tongue), buccal mucosa, gingiva, mouth bottom and hard palate, is one of the more common malignant tumors of the sixth malignant tumor well-developed area (head and neck) of the world, is second to nasopharyngeal carcinoma and is located at the second place of the head and neck malignant tumors. Oral cancer can occur at any age, with a peak in onset at 40-60 years of age, with a ratio of about 2:1 for both men and women. In recent years, oral cancer has exhibited the following new characteristics: firstly, the growth rate of the disease rate of tongue cancer is faster and is close to half of all oral cancer; secondly, the sick age of the oral cancer is in a trend of youthfulness, and young patients of 20-30 years old are not rare; third, female patients increase year by year. In the past 20 years, although traditional treatment methods including surgery, radiotherapy and chemotherapy are developed to different degrees, the prognosis of oral cancer is still unsatisfactory, the 5-year survival rate is only about 50-60%, about 1/3 of patients can relapse, swallowing, speech function, facial appearance and the like of patients treated by surgery are seriously affected, and obvious and serious facial injuries, eating and language disorders exist. The main causes affecting survival are local recurrence and lymphatic metastasis. Therefore, new therapeutic approaches such as biological therapeutic approaches like targeted tumor therapy are receiving increasing attention. Meanwhile, how to improve the anticancer efficacy of drug therapy and reduce the toxic and side effects of the drug, namely the targeting of the anticancer therapy, is a concern for the antitumor basis and clinical research.

Two-dimensional cell culture is mostly adopted in the traditional clinical drug sensitivity detection. However, the two-dimensional cultured cells only simulate the tissue physiological conditions to a limited extent, lack the real tissue structure in vivo, easily cause low differentiation level and loss of cell physiological functions, and further cause the obtained experimental results to be difficult to predict the clinical practical results. Organoids, belonging to three-dimensional (3D) cell cultures, are mainly derived from human embryonic stem cells, induced pluripotent stem cells and adult stem cells that have differentiation capacity. Endogenous tissue stem cells exist in different tissues and organs, and play an important role in maintaining the functional forms of the organs. Under certain inducing conditions in vitro, the stem cells can self-organize to form a mini-structure with the diameter of only a few millimeters. Tumor organoids are miniature 3D tumor cell models grown in the laboratory from primary tumors taken from patients. The tumor organoids highly mimic the characteristics of the source tumor tissue, retain inter-individual tumor heterogeneity, and can be used for functional testing, such as high-throughput drug screening and individualized precision therapy.

At present, basal medium (DMEM or DMEM/F12), R-spondin-1, noggin and some expensive protein factors are mostly adopted in the oral cancer organoid culture method, so that organoid culture cost is high; and the technical operation is complex and the technical difficulty is high, so that the large-scale commercial application of the technology is limited. Therefore, there is a need to develop a low-cost, simple organoid culture method and medium with high success rate.

Disclosure of Invention

In order to solve the technical problems, the invention provides a culture medium and a culture method for rapidly amplifying oral cancer organoids in vitro.

One aspect of the invention is to provide a culture medium for an oral cancer organoid, comprising an MST1/2 kinase inhibitor, at least one cell culture additive selected from N2 and B27, a fibroblast growth factor, CHIR99021, an epidermal growth factor, insulin, an ITS cell culture additive, SB202190, Y27632, dexamethasone, glutaMAX, and non-essential amino acids. Wherein the MST1/2 kinase inhibitor comprises a compound of formula (I) or a pharmaceutically acceptable salt, or solvate thereof,

wherein the content of the first and second substances,

R ₁ selected from C1-C6 alkyl, C3-C6 cycloalkyl, C4-C8 cycloalkylalkyl, C2-C6 spirocycloalkyl, and optionally substituted by 1-2 independent R ₆ Substituted aryl (e.g., phenyl, naphthyl, and the like), arylC 1-C6 alkyl (e.g., benzyl, and the like), and heteroaryl (e.g., thienyl, and the like);

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

R ₄ and R ₅ Each independently selected from hydrogen, C1-C6 alkyl, C3-C6 cycloalkyl, C4-C8 cycloalkylalkyl, C1-C6 alkylhydroxy, C1-C6 haloalkyl, C1-C6 alkylaminoC 1-C6 alkyl, C1-C6 alkoxyC 1-C6 alkyl, and C3-C6 heterocycloC 1-C6 alkyl (said heterocyclyl is selected from, for example, piperidinyl, tetrahydropyranyl, and the like);

R ₆ selected from the group consisting of 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,

R ₁ selected from C1-C6 alkyl, optionally substituted with 1-2 independent R ₆ Substituted phenyl, optionally substituted with 1-2 independent R ₆ Substituted thienyl, and optionally substituted with 1-2 independent R ₆ Substituted benzyl, R ₁ More preferably optionally substituted with 1-2 independent R ₆ Substituted phenyl;

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

R ₆ each independently selected from halogen, C1-C6 alkyl, and C1-C6 haloalkyl, R ₆ More 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 content of each component in the medium of the invention satisfies any one or more or all of the following:

(1) The concentration of the MST1/2 kinase inhibitor is preferably 2.5-10 mu M;

(2) The volume ratio of the B27 or N2 cell culture additive to the culture medium is 1;

(3) The concentration of the fibroblast growth factor is preferably 3-30 ng/mL;

(4) The concentration of CHIR99021 is preferably 1.25-5 μ M;

(5) The concentration of the epidermal growth factor is preferably 2-18 mu M;

(6) The concentration of the insulin is preferably 1 to 10 mug/mL;

(7) The volume ratio of the ITS cell culture additive to the culture medium is preferably 1;

(8) The concentration of SB202190 is preferably 100-400 nM;

(9) The concentration of Y27632 is preferably 3-30 μ M;

(10) The concentration of dexamethasone is preferably 0.02-0.5 mu M;

(11) The volume ratio of GlutaMAX to the medium is preferably 1;

(12) The nonessential amino acid is one or more selected from glycine, alanine, asparagine, aspartic acid, glutamic acid, proline and serine, and the concentration of the nonessential amino acid is preferably 50 to 200. Mu.M.

In an embodiment of the invention, said medium further comprises a starting medium selected from DMEM/F12, DMEM, F12 or RPMI-1640; and an antibiotic selected from one or more of streptomycin/penicillin, amphotericin B, and Primocin.

In a preferred embodiment, the concentration of streptomycin ranges from 25 to 400. Mu.g/mL when the antibiotic is selected from streptomycin/penicillin, the concentration of penicillin ranges from 25 to 400U/mL, the concentration ranges from 0.25 to 4. Mu.g/mL when the antibiotic is selected from amphotericin B, and the concentration ranges from 25 to 400. Mu.g/mL when the antibiotic is selected from Primocin.

The invention also provides a culture method of the oral cancer organoid. In the method for culturing an oral cancer organoid of the present invention, the oral cancer organoid is cultured using the oral cancer organoid culture medium of the present invention.

The oral cancer organoid culture method of the present invention comprises the following steps.

1. And (3) separating a sample from the oral cancer solid tumor tissue to obtain oral cancer primary cells. The treatment process comprises the following steps:

(1) Separating oral cancer tissue samples, adding a base culture medium and tissue digestive juice (the adding amount of the tissue digestive juice is about 10mL per 1g of tumor tissue) in proportion of 1:3, placing the mixture in a constant temperature shaking table for digestion, wherein the digestion temperature is 4-37 ℃, the rotation speed of the shaking table is 200-350 rpm, and the digestion time is 3-6 hours;

(2) After digestion, centrifuging and discarding supernatant, wherein the centrifugation speed is 1200-1600 rpm, and the centrifugation time is 2-6 minutes.

Wherein the basal medium formulation comprises an initial medium selected from DMEM/F12, DMEM, F12 or RPMI-1640; and an antibiotic selected from one or more of streptomycin/penicillin, amphotericin B, and Primocin. The formula of the tissue digestive fluid comprises a 1640 culture medium, collagenase II (1-2 mg/mL), collagenase IV (1-2 mg/mL), DNase (50-100U/mL), hyaluronidase (0.5-1 mg/mL), calcium chloride (1-5 mM) and bovine serum albumin BSA (5-10 mg/mL).

2. Preparing the oral cancer organoid culture medium, and culturing the oral cancer primary cells obtained in the step.

Resuspending and counting the oral cancer primary cells obtained in the step 1 by using the oral cancer organoid culture medium of the invention, and diluting the cell density to 5-10 × 10 ⁶ And (2) taking out the diluted cell suspension, adding the diluted cell suspension into Matrigel with the same volume, uniformly mixing, then inoculating the mixture into a porous plate, putting the inoculated porous plate into an incubator for 30-60 minutes until Matrigel is completely solidified, and then adding an oral cancer organoid culture medium for expanded culture.

The present invention also provides a method for evaluating or screening a drug for treating oral cancer diseases, comprising the steps of:

(1) Culturing the oral cancer organoids by using the culture method of the oral cancer organoids of the present invention;

(2) Selecting a medicine to be detected and diluting according to a required concentration gradient;

(3) Adding the diluted medicine to the organoid cultured in (1);

(4) Organoid size or organoid viability testing is performed.

The beneficial effects of the invention include:

(1) The success rate of culturing the oral cancer organoid is improved and reaches over 90 percent;

(2) Ensuring that the primarily cultured oral cancer organoids in vitro can maintain the pathological characteristics of the patient;

(3) The amplification efficiency is high, the oral cancer organoids can be quickly cultured, and the amplified oral cancer organoids can be continuously passed;

(4) The culture cost is controllable, and expensive factors such as Wnt agonist, R-spondin family protein, noggin protein, BMP inhibitor, fibroblast growth factor 10 (FGF 10) and the like do not need to be added into the culture medium;

(5) The oral cancer organoids obtained by the culture of the technology have large quantity, and are suitable for screening candidate compounds in high flux and providing high-flux drug in-vitro sensitivity function tests for patients.

Drawings

FIGS. 1A-1L are graphs showing the effect of different concentrations of factors added to oral cancer organoid media of the present invention on oral cancer organoid proliferation.

FIGS. 2A-2D are photographs of observing, with a microscope, oral cancer organoids cultured using the oral cancer organoid medium of the present invention, wherein FIG. 2A shows photographs of organoids obtained by culturing a sample OZ-018 after 8 days; FIG. 2B shows a photograph of organoids obtained by OZ-019 culture of a specimen after 10 days; FIG. 2C shows a photograph of organoids obtained from the culture of sample OZ-020 after 7 days; FIG. 2D shows a photograph of organoids cultured from sample OZ-021 after 8 days.

FIG. 3A shows the results of pathological and immunohistochemical identification of oral cancer organoids cultured with the oral cancer organoid medium of the present invention on sample OZ-020; FIG. 3B shows the results of pathological and immunohistochemical identification of OZ-020 tissue in a sample; FIG. 3C shows the results of pathological and immunohistochemical identification of oral cancer organoids obtained by culturing a sample OZ-021 in an oral cancer organoid culture medium according to the present invention; FIG. 3D shows the results of pathological and immunohistochemical characterization of OZ-021 tissues in the samples.

FIG. 4 is a comparison of the culture of oral cancer organoids using the oral cancer organoid medium of the present invention and a conventional culture medium, wherein FIG. 4A shows photographs after 10 days of culture with the OC-3 medium of the present invention; FIG. 4B shows a photograph after 10 days of culture with ED medium; FIG. 4C shows a histogram comparing the relative sizes of organoids cultured in OC-3 medium and ED medium.

FIG. 5 shows the results of different drug sensitivity tests on oral cancer organoids cultured using the oral cancer organoid medium of the present invention, wherein FIG. 5A shows photographs of organoids grown without drug treatment and after 3 days of drug treatment; figure 5B shows a bar graph of the inhibition rate of various concentrations of test drug to inhibit oral cancer organoid growth.

Detailed Description

For a better understanding of the present invention, the present invention is further described below with reference to the following examples and the accompanying drawings. The following examples are intended to illustrate the invention without limiting it.

[ preparation example of MST1/2 kinase inhibitor ]

In the present specification, an MST1/2 kinase inhibitor refers to any inhibitor that directly or indirectly down-regulates MST1/2 signaling. In general, MST1/2 kinase inhibitors, 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 1 4- ((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 (A2): after 2-amino-2- (2,6-difluorophenyl) acetic acid (2.0 g) was added to the round bottom flask, methanol (30 ml) was added followed by thionyl chloride (1.2 ml) dropwise under ice-bath. The reaction system was reacted at 85 ℃ overnight. After the reaction was completed, the solvent was evaporated to dryness under reduced pressure to obtain a white solid, which was used in the next step.

Methyl 2- ((2-chloro-5-nitropyrimidin-4-yl) amino) -2- (2,6-difluorophenyl) acetate (A3): to a round bottom flask was added methyl 2-amino-2- (2,6-difluorophenyl) acetate (2 g) followed by acetone (30 ml) and potassium carbonate (2.2 g) then the system was cooled to-10 ℃ with an ice salt bath followed by the slow addition 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 (A4): to a round bottom flask was added methyl 2- ((2-chloro-5-nitropyrimidin-4-yl) amino) -2- (2,6-difluorophenyl) acetate (2.5 g) followed by acetic acid (50 ml) and iron powder (3.9 g). The reaction 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 a compound A4.LC/MS: m + H297.0.

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 flask, cooled to-35 deg.C, iodomethane (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 a 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 was added 2-chloro-7- (2,6-difluorophenyl) -5,8-dimethyl-7,8-dihydropteridin-6 (5H) -one (100 mg), sulfanilamide (53 mg), p-toluenesulfonic acid (53 mg), and sec-butanol (5 ml). The reaction was stirred at 120 ℃ 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 Effect of factors added to oral cancer organoid Medium on proliferation of oral cancer organoids

(1) Preparation of oral cancer organoid culture medium

First, a basal medium containing an initial medium is prepared. The starting medium may be selected from DMEM/F12, DMEM, F12 or RPMI-1640 commonly used in the art. In this example, the basic medium formulation is: DMEM/F12 medium (from Corning) + 100. Mu.g/mL Primocin (from InvivoGen, 0.2% (v/v), commercial product concentration 50 mg/mL).

Different additives (see table 1) are added into the basic culture medium respectively to prepare the oral cancer organoid culture medium containing different additive components.

(2) Isolation and treatment of primary oral cancer cells

1 sample selection

Oral cancer solid tumor tissue samples (intraoperatively) were obtained from patients who had signed informed consent by medical professionals at a medical professional. Intraoperative sample 0.25cm ³ Commercial tissue preservation solution (manufacturer:miltenyi Biotec).

2 Material preparation

And (3) sterilizing the surfaces of a 15mL sterile centrifuge tube, a pipette, a 10mL pipette, a sterile gun head and the like, and then placing the sterilized surfaces into an ultra-clean workbench for ultraviolet irradiation for 30 minutes. Basal medium DMEM/F12 (Corning) was removed from the 4 ℃ freezer 30 minutes earlier and tissue digestate was removed from the-20 ℃ freezer 30 minutes earlier.

The formula of the tissue digestive juice comprises the following components: 1640 medium (Corning, 10-040-CVR), collagenase II (2 mg/mL), collagenase IV (2 mg/mL), DNase (50U/mL), hyaluronidase (0.75 mg/mL), calcium chloride (3.3 mM), bovine serum albumin BSA (10 mg/mL).

Collagenase ii, collagenase iv, dnase, hyaluronidase mentioned above were all purchased from Sigma company; calcium chloride was purchased from bio-engineering (shanghai) gmbh; BSA was purchased from Biofrox.

3 separation of samples

3.1 taking tissue samples in a Petri dish in a clean bench, removing blood-carrying tissue, rinsing 2 times with a basal medium, transferring the tissue to another Petri dish, mechanically separating with a sterile scalpel, dividing the tissue block into 1 x 1mm ³ Size;

3.2 sucking the cut intraoperative tissue into a 15mL centrifuge tube, adding 5mL basic culture medium, uniformly mixing, and centrifuging at 1500rpm for 4 minutes;

3.3 abandoning the supernatant, adding the basal medium and the tissue digestive juice in a proportion of 1:3 (note: the adding amount of the tissue digestive juice is 1g of tumor tissue and about 10mL of the tissue digestive juice), marking the name and the number of the sample, sealing the sample by a sealing film, digesting the sample in a shaking table (ZQLY-180N) at 300rpm at 37 ℃, observing whether the digestion is finished every 30 minutes, and judging that no visible particles exist;

3.4 after the digestion is finished, filtering out undigested tissue agglomerates by a 100-micron filter screen, flushing the tissue agglomerates on the filter screen into a centrifuge tube by using a basic culture medium to reduce cell loss, and centrifuging at 1500rpm for 4 minutes at 25 ℃;

3.5 abandoning the supernatant, observing whether blood cells exist, if the blood cells exist, adding 8mL of blood cell lysate (purchased from Sigma company), mixing uniformly, cracking at 4 ℃ for 20 minutes, reversing and mixing uniformly for once, and centrifuging at 25 ℃ at 1500rpm for 4 minutes;

3.6 discard the supernatant, add 2mL of basal medium to resuspend the cells for use.

4 cell counting and processing

4.1 Observation under a lens: a small amount of the resuspended cells are removed and spread in a culture dish, and the density and morphology of the cancer cells are observed under a microscope (CNOPTEC, BDS 400);

4.2 viable cell count: after 12. Mu.L of the resuspended cell suspension and 12. Mu.L of trypan blue stain (manufacturer: biotechnology (Shanghai) Co., ltd.) were mixed well, 20. Mu.L of the mixture was added to a cell counting plate (manufacturer: countstar, specification: 50 plates/cassette), and the percentage of viable large cells (cell size >10 μm) was calculated using a cell counter (Countstar, IC 1000) = viable cell count/total cell count of 100%.

(3) Culture of oral cancer organoids

Resuspending and counting the oral cancer primary cells obtained in the step by using precooled DMEM/F12, and diluting the cell density to 5-10 multiplied by 10 ⁶ mu.L/mL of the diluted cell suspension was taken out and added to an equal volume of Matrigel (Corning) and gently mixed, and then the mixture was seeded in a 96-well plate at 5. Mu.L/well. The inoculated culture plate was placed in an incubator for 30 minutes until Matrigel was completely solidified, then the culture media shown in table 1, which had been restored to room temperature in advance, were added, respectively, and the culture media were changed every three days for the expanded culture. The cultured organoids were photographed 7 days later, and the diameters of the organoids were measured and counted, and the promotion effects of the factors on the proliferation of oral cancer organoids were compared. Among them, as an experimental control, a basal medium without any additive was used, and the experimental results are shown in table 1.

TABLE 1 additional ingredients in culture media and organoid proliferation promoting effects

Serial number	Media additive species	Suppliers of goods	Final concentration	Grading of proliferation promoting degree
					1	N2	Gibco	1:50	+
2	EGF	R&D	5ng/mL	+
					3	R-spondin1	R&D	20ng/mL	+
4	Prostaglandin E2	Tocris	0.5μM	○
					5	Insulin	Peprotech	1.5μg/mL	+
6	B27	Gibco	1:50	+
					7	A8301	MCE	100nM	-
8	SB202190	MCE	200nM	+
					9	bFGF	R&D	10ng/mL	+
10	Hydrocortisone	Sigma	10ng/mL	○
					11	Noggin	R&D	30ng/mL	○
12	FBS	Excell		5％						+
					13	IGF-1	R&D	45ng/mL	○
14	KGF	R&D	5ng/mL	○
					15	GlutaMAX	Gibco	1:100	+
16	Non-essential amino acids	Corning	100μM	+
					17	Dexamethasone	MCE	0.1μM	+
18	NRG1	sino biological	5ng/mL	○
					19	Y27632	MCE	10μM	+
20	ITS	Gibco	1:100	+
					21	Compound 1	Preparation example	5μM	+
22	CHIR99021	MCE	2.5μM	+

Wherein "+" indicates that the culture medium added with the additive has proliferation promoting effect on at least two of oral cancer organoids separated from oral cancer tissues compared with basal culture medium; "-" indicates that the medium to which the additive was added showed an inhibitory effect on the proliferation of at least one of oral cancer organoids isolated from oral cancer tissues; ". Smallcircle" indicates that the medium to which the additive was added had no significant effect on the proliferation of at least two of the oral cancer organoids isolated from oral cancer tissues.

According to the results, factors such as B27, fibroblast growth factor bFGF, CHIR99021, epidermal growth factor EGF, insulin, ITS cell culture additives, SB202190, Y27632, dexamethasone, glutaMAX, compound 1, non-essential amino acids and the like are selected for further culture experiments.

EXAMPLE 2 proliferation of oral cancer organoids by Medium supplementation with different concentrations of factors

Oral cancer primary cells were obtained from intraoperative tissue samples (nos. OZ013 and OZ 014) according to the method of (2) of example 1, and organoid culture was performed using the procedure in table 2 below.

TABLE 2 Medium composition (final concentration)

When the culture medium of formula 1 is used, 200 μ L of B27 prepared per well is added to a 96-well plate inoculated with organoids on the basis of formula 1, and the final concentrations of B27 are 1; and a control well (BC) was set using the medium of formulation 1. The final concentrations of the other additional factors in the series of media were the same as in OC-3 media. The following experiments for formulations 1 to 12 were also performed in the same manner and will not be described in detail.

When the culture medium of the formula 2 is used, 200 mu L of prepared bFGF is added into a 96-well plate inoculated with organoids on the basis of the formula 2, wherein the final concentration of the bFGF is 3ng/mL, 10ng/mL and 30ng/mL respectively; and control wells (BC) were set using medium of formula 2.

When the culture medium of the formula 3 is used, 200 mu L of prepared CHIR99021 is added into a 96-well plate inoculated with an organoid on the basis of the formula 3, wherein the final concentration of the CHIR99021 is 1.25 mu M, 2.5 mu M and 5 mu M respectively; and control wells (BC) were set using the medium of formulation 3.

When the culture medium of the formula 4 is used, 200 mu L of the prepared EGF is added into a 96-well plate inoculated with an organoid on the basis of the formula 4, wherein the final concentration of the EGF is 2ng/mL, 6ng/mL and 18ng/mL respectively; and control wells (BC) were set using medium of formula 4.

When the culture medium of the formula 5 is used, 200 mu L of prepared insulin is added into a 96-well plate inoculated with an organoid on the basis of the formula 5, wherein the final concentrations of the insulin are 1 mu g/mL, 3 mu g/mL and 10 mu g/mL respectively; and control wells (BC) were set using medium of formulation 5.

When the culture medium of formula 6 is used, 200 μ L of prepared ITS per well is added to a 96-well plate inoculated with organoids on the basis of formula 6, and the final concentration of ITS is 1; and control wells (BC) were set using medium of formula 6.

When the culture medium of formula 7 is used, 200 μ L of prepared SB202190 is added to each well of formula 7 in a 96-well plate inoculated with organoids, and the final concentrations of SB202190 are 100nM, 200nM and 400nM, respectively; and control wells (BC) were set using medium of formula 7.

When the culture medium of the formula 8 is used, 200 mu L of prepared Y27632 is added into a 96-well plate inoculated with organoids on the basis of the formula 8, and the final concentration of Y27632 is 3 mu M, 10 mu M and 30 mu M respectively; and control wells (BC) were set using medium of formula 8.

When the culture medium of the formula 9 is used, 200 mu L of prepared dexamethasone is added into a 96-well plate inoculated with organoids on the basis of the formula 9, wherein the final concentrations of the dexamethasone are 0.02 mu M, 0.1 mu M and 0.5 mu M respectively; and control wells (BC) were set using medium of formulation 9.

When the culture medium of formula 10 is used, 200 μ L of prepared GlutaMAX per well is added to a 96-well plate inoculated with organoids on the basis of formula 10, and the final concentrations of GlutaMAX are 1; and control wells (BC) were set using medium of formula 10.

When the culture medium of formula 11 is used, 200. Mu.L of the prepared compound 1 per well is added to a 96-well plate inoculated with organoids on the basis of formula 11, and the final concentrations of the compound 1 are 2.5. Mu.M, 5. Mu.M and 10. Mu.M, respectively; and control wells (BC) were set using medium of formula 11.

When the culture medium of the formula 12 is used, 200 mu L of the prepared non-essential amino acid is added into a 96-well plate inoculated with an organoid on the basis of the formula 12, wherein the final concentration of the non-essential amino acid is 50 mu M, 100 mu M and 200 mu M respectively; and control wells (BC) were set using medium of formula 12.

After 7 days, the cultured organoids were photographed, and the sizes of diameters of the organoids were measured and counted to compare the promoting effects of the concentrations of the factors on the proliferation of the oral cancer organoids. The data collected for 2 samples are summarized in FIGS. 1A to 1L. In FIGS. 1A-1L, the ratio is the ratio of the organoid diameter obtained by 7 days of culture using each medium to the organoid diameter obtained by 7 days of culture in the corresponding BC control wells. 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 Kong Peiyang 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.

From the results of fig. 1A to 1L, the volume concentration of B27 is preferably 1; the content of fibroblast growth factor bFGF is preferably 3-30 ng/mL; the content of CHIR99021 is preferably 1.25-5 μ M; the content of the epidermal growth factor EGF is preferably 2-18 ng/mL; the content of the insulin is preferably 1-10 mug/mL; the volume concentration of the ITS cell culture additive is preferably 1; the content of SB202190 is preferably 100-400 nM; the content of Y27632 is preferably 3-30 mu M; the content of dexamethasone is preferably 0.02-0.5 mu M; the volume concentration of GlutaMAX is preferably 1; the content of the MST1/2 kinase inhibitor is preferably 2.5-10 mu M; the content of the non-essential amino acid is preferably 50 to 200. Mu.M.

Example 3 oral cancer organoid culture and identification

The oral cancer primary cells obtained as described in (2) of example 1 were resuspended and counted in the oral cancer organoid culture medium OC-3 of the present invention, and the cell density was diluted to 5 to 10X 10 ⁶ Per mL, 400. Mu.L of the diluted cell suspension was removed and added to an equal volume of Matrigel (Corning)Mix well and then inoculate the mixture in 24-well plates at 50. Mu.L/well. The inoculated culture plate is put into an incubator for 30 minutes until Matrigel is completely solidified, then oral cancer organoid culture medium OC-3 which is restored to room temperature in advance is added, 500 mu L of the culture medium is added into each hole, and the culture medium is replaced once every three days for amplification culture.

The cultured oral cancer organoids were observed on days 7 to 10 using a microscope (EVOS M500, invitrogen). FIGS. 2A-2D are photographs of oral cancer organoids obtained by culturing OZ-018, OZ-019, OZ-020 and OZ-021 samples under a 10-fold objective lens. The oral cancer organoids are regular spheres under the mirror, and the surface is smooth.

And performing pathological and immunohistochemical identification on the oral cancer organoids obtained by culture, simultaneously sending corresponding tissue samples to perform pathological and immunohistochemical identification, and comparing the consistency of organoids and tissue results.

FIGS. 3A and 3C are the results of pathological and immunohistochemical identification of oral cancer organoids obtained by in vitro culture of OZ-020 and OZ-021 samples, which are pictures photographed under a 20-fold objective lens. As shown in fig. 3A and 3C, HE results show that the organoid structural morphology is cancer tissue morphology; CK. 34 beta E12, P63, CK5/6, ki67 expression, suggesting that the sample is oral cancer. FIGS. 3B and 3D show the pathological and immunohistochemical results of tissues corresponding to OZ-020 and OZ-021, and the results show that the oral cancer organoids cultured by using the culture medium OC-3 of the invention are consistent with the diagnosis results of oral cancer tissues.

Example 4 comparison of culture Effect with existing Medium

(1) Preparation of control Medium

Preparation of the medium used in the literature (Else Driehuis et al, cancer Discov.2019, 9. Hereinafter referred to as ED medium.

(2) Oral cancer organoid culture

Oral cancer primary cells were obtained from intraoperative tissue sample OZ-022 according to the method of (2) of example 1, and organoid culture was performed according to the method of example 3 using OC-3 medium and ED medium, respectively.

On the 10 th day of culture, the cultured oral cancer organoids were observed with a microscope (EVOS M500, invitrogen). FIGS. 4A and 4B are photographs taken of organoids cultured with OC-3 medium and ED medium, respectively, under a 10-fold objective lens, and FIG. 4C is a bar graph comparing the relative sizes of organoids cultured with the two media.

From the results of FIGS. 4A-4C, it can be seen that OC-3 medium significantly promoted the expansion and culture of oral cancer organoids compared to ED medium.

Example 5 oral cancer organoids amplified using the culture Medium of the present invention for drug screening

(1) Oral cancer organoid culture

From the obtained intraoral cancer intraoperative sample (OZ-016), oral cancer primary cells were isolated according to the method of (2) of example 1, and organoid culture was performed using OC-3 medium, and drug screening was performed when the diameter of oral cancer organoid exceeded 50 μm.

(2) Screening drug formulations

3 drugs (daunorubicin, aclarubicin, doxorubicin; all from MCE) were formulated at 2 concentration gradients according to the following table and stored for later use.

TABLE 3 preparation of daunorubicin, aclarubicin, doxorubicin drug additive solution

Daunorubicin	Final concentration (μ M)	1	0.1
				Acrubicin	Final concentration (μ M)	1	0.1
Doxorubicin	Final concentration (μ M)	0.1	0.01

(3) Dosing

Taking out the prepared medicine, placing at room temperature, and diluting the medicine 1000 times with OC-3 culture medium for later use. Taking out the organoid cultured according to the step (1) from the incubator, removing the culture medium in the culture well, and slowly introducing the culture medium containing the drug into the culture well along the wall of the culture well. After the medicine is added, the surface of the 96-pore plate is disinfected and then is moved to an incubator to be cultured continuously, and after 72 hours, the organoid activity is measured.

(4) Organoid viability assay

CellTiter-Glo luminescence reagent (purchased from Promega) was taken out from a refrigerator at 4 ℃,10 ml of the reagent was put into a sample addition tank, a 96-well plate to be tested was taken out from an incubator, 20. Mu.L of CellTiter-Glo luminescence reagent was added to each well, and the mixture was left to stand for 10 minutes and then mixed, and was subjected to detection using a multifunctional microplate reader (Envision, perkin Elmer Co.).

(5) Data processing

Drug inhibition rate (%) =100% - (culture well chemiluminescence value on third day) according to the formula _{Drug treatment group} Culture on day zeroHole chemiluminescence values _{Drug treatment group} ) /(day three culture wells chemiluminescence values _DMSO Chemiluminescence values of culture wells on day zero _DMSO ) 100%, inhibition rates of different drugs were calculated and the results are shown in fig. 5A and 5B. Fig. 5A is a photograph of organoid growth without drug treatment and 3 days after drug treatment taken with a 4-fold objective microscope (EVOS M500, invitrogen) and fig. 5B is a bar graph of the rate of inhibition of growth of oral cancer organoids by test drugs at different concentrations.

From FIG. 5A, it was confirmed that the organoid growth state obtained by the culture using the oral cancer organoid medium of the present invention was good, and the organoid growth was significantly inhibited after the treatment with daunorubicin and doxorubicin. Figure 5B is a bar graph of the inhibition rate of three test drugs at different concentrations for inhibiting oral cancer organoid growth. As can be seen in FIG. 5B, the data error for the drug treatment group was small, indicating that the data remained substantially consistent between duplicate wells of the same drug when the jacket system was used for drug screening. Among the three antitumor drugs, aclarubicin has a strong effect of inhibiting organoid growth under two concentrations, the differences of the inhibition effects of daunorubicin with different concentrations are obvious, and the inhibition effect of doxorubicin on organoid growth is weak, which indicates that the organoids of the same patient have different sensitivities to different drugs. According to the result, the effectiveness and the effective dosage of the medicine can be judged when the oral cancer patient uses the medicine clinically.

Claims (10)

1. A culture medium for an oral cancer organoid comprising an MST1/2 kinase inhibitor, at least one cell culture additive selected from N2 and B27, fibroblast growth factor, CHIR99021, epidermal growth factor, insulin, ITS cell culture additive, SB202190, Y27632, dexamethasone, glutaMAX, and a non-essential amino acid,

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

wherein, the first and the second end of the pipe are connected with each other,

R ₁ selected from C1-C6 alkyl, C3-C6 cycloalkyl, C4-C8 cycloalkylalkyl, C2-C6 spirocycloalkyl, and optionally substituted with 1-2 independent R ₆ Substituted aryl, arylC 1-C6 alkyl and heteroaryl;

R ₂ and R ₃ Each independently selected from C1-C6 alkyl;

R ₄ and R ₅ Each independently selected from hydrogen, C1-C6 alkyl, C3-C6 cycloalkyl, C4-C8 cycloalkylalkyl, C1-C6 alkylhydroxy, C1-C6 haloalkyl, C1-C6 alkylaminoC 1-C6 alkyl, C1-C6 alkoxyC 1-C6 alkyl, and C3-C6 heterocycloC 1-C6 alkyl;

R ₆ selected from the group consisting of halogen, C1-C6 alkyl, C1-C6 alkoxy, and C1-C6 haloalkyl.

2. The culture medium of claim 1, wherein

R ₁ Selected from C1-C6 alkyl, C3-C6 cycloalkyl, C4-C8 cycloalkylalkyl, C2-C6 spirocycloalkyl, and optionally substituted with 1-2 independent R ₆ Substituted phenyl, naphthyl, benzyl and thienyl;

R ₂ and R ₃ Each independently selected from C1-C3 alkyl;

R ₄ and R ₅ Each independently selected from hydrogen, C1-C6 alkyl, C3-C6 cycloalkyl, C4-C8 cycloalkylalkyl, C1-C6 alkylhydroxy, C1-C6 haloalkyl, C1-C6 alkylaminoC 1-C6 alkyl, C1-C6 alkoxyC 1-C6 alkyl, piperidinylC 1-C6 alkyl, and tetrahydropyranyl C1-C6 alkyl;

R ₆ selected from the group consisting of halogen, C1-C6 alkyl, C1-C6 alkoxy, and C1-C6 haloalkyl.

3. The 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,

R ₁ selected from C1-C6 alkyl, optionally substituted by 1-2 independent R ₆ Substituted phenyl, optionally substituted with 1-2 independent R ₆ Substituted thienyl, and optionally substituted with 1-2 independent R ₆ A substituted benzyl group;

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

R ₆ each independently selected from halogen, C1-C6 alkyl, and C1-C6 haloalkyl.

4. The culture medium of claim 3, wherein

R ₁ Is optionally substituted by 1-2 independent R ₆ Substituted phenyl;

R ₅ is hydrogen;

R ₆ preferably fluorine, methyl or trifluoromethyl.

5. The 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. a culture medium according to any one of claims 1 to 5, wherein the content of each component in the culture medium satisfies any one or more or all of the following:

the concentration of the MST1/2 kinase inhibitor is 2.5-10 mu M;

the volume ratio of the B27 or N2 cell culture additive to the culture medium is 1;

the concentration of the fibroblast growth factor is 3-30 ng/mL;

the concentration of the CHIR99021 is 1.25-5 mu M;

the concentration of the epidermal growth factor is 2-18 mu M;

the concentration of the insulin is 1-10 mug/mL;

the volume ratio of the ITS cell culture additive to the culture medium is 1;

the concentration of the SB202190 is 100-400 nM;

the concentration of the Y27632 is 3-30 mu M;

the concentration of the dexamethasone is 0.02-0.5 mu M;

the volume ratio of the GlutaMAX to the culture medium is 1;

the non-essential amino acid is one or more selected from glycine, alanine, asparagine, aspartic acid, glutamic acid, proline and serine, and the concentration of the non-essential amino acid is 50-200 mu M.

7. The culture medium according to any one of claims 1 to 5, further comprising:

a starting medium selected from DMEM/F12, DMEM, F12 or RPMI-1640; and

an antibiotic selected from one or more of streptomycin/penicillin, amphotericin B and Primocin.

8. The culture medium according to any one of claims 1 to 5, wherein said medium is free of Wnt agonists, R-spondin family proteins, noggin proteins, BMP inhibitors, fibroblast growth factor 10.

9. A method for culturing an oral cancer organoid, comprising the steps of:

(1) Separating a sample from oral cancer solid tumor tissues to obtain oral cancer primary cells;

(2) Preparing a culture medium for an oral cancer organoid according to any of claims 1-8 and subjecting the oral cancer primary cells obtained in step (1) to organoid culture.

10. A method for evaluating or screening a drug for treating an oral cancer disease, comprising the steps of:

(1) Culturing an oral cancer organoid using the method of culturing an oral cancer organoid of claim 9;

(2) Selecting a medicine to be detected and diluting according to a required concentration gradient;

(3) Adding the diluted medicine to the organoid cultured in (1);

(4) Organoid size or organoid viability assays are performed.

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