CN115975937A - Culture medium and culture method for liver cancer suspension organoid - Google Patents

Abstract

The invention relates to a culture medium for liver cancer suspension organoid culture, which comprises an MST1/2 kinase inhibitor, Y27632, at least one cell culture additive selected from N2 and B27, SB202190, insulin, fibroblast growth factor 10, cholera toxin, ITS cell culture additives, hepatocyte growth factor, glutaMAX and non-essential amino acids. The invention also relates to a culture method and application of the liver cancer suspension organoid. By using the liver cancer suspension organoid culture medium, the liver cancer cells can be effectively and rapidly amplified, so that the liver cancer cells obtained by amplification keep the pathological characteristics of patients, the culture success rate and the amplification rate of the liver cancer cells are improved, and a research basis can be provided for the personalized treatment of the patients.

Description

Culture medium and culture method for liver cancer suspension organoid

Technical Field

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

Background

Liver cancer is the third most common malignant tumor with mortality rate second to gastric cancer and esophageal cancer. In 2020, more than 90 thousands of people newly diagnosed with liver cancer and more than 83 thousands of people died due to liver cancer are in the world, and the number of the died people is close to the number of new diseases.

In recent years, adjuvant chemotherapy for liver cancer after operation has been paid more attention and accepted by clinicians as a new adjuvant therapy, including postoperative TACE therapy, oral drug therapy, etc., but because of lack of standard chemotherapy schemes, conventional chemotherapy based on experience ignores individual differences and has certain blindness, the effect is always poor, and the effective rate of single drug and combined drug is less than 20% (Jendal A, thadi A, shailubhai K.hepatocellular Carcinoma: iology and Current and Future Drugs [ J ]. J Clin Exp Hepatol,2019,9 (2): 221-232). Although the toxic and side effects of emerging targeted drugs are reduced to a certain extent, the number of the emerging targeted drugs is too small, the treatment cost is high, the effective rate varies with individuals, and the treatment requirements of most patients are difficult to meet. Because an effective liver cancer drug sensitivity test system is lacked, accurate chemotherapy cannot be realized, so that the correspondence of the in vitro drug sensitivity result of the liver cancer and the clinical in vivo reaction becomes the key treatment.

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. The organoids are mainly derived from embryonic stem cells, induced pluripotent stem cells and adult stem cells which have the differentiation capacity of the human body. Endogenous tissue stem cells exist in different tissues and organs, and play an important role in maintaining the functional morphology of each organ. Under certain induction conditions in vitro, the stem cells can self-organize to form a mini-structure with a diameter of only a few millimeters. Tumor organoids are micro 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.

Currently, the liver cancer organoid culture method mostly adopts R-spondin-1, noggin and some expensive protein factors, so that the organoid culture cost is high; the technology needs to use matrigel to carry out coating treatment in advance, and has complex culture and passage operation and great technical difficulty; due to the coating of matrigel, the cost of large-scale culture is high, the standardization difficulty is high, and the large-scale commercial application is limited. Therefore, there is a need to develop a low-cost, simple and high-success organoid culture method and medium.

Disclosure of Invention

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

One aspect of the present invention provides a liver cancer suspension organoid culture medium comprising an MST1/2 kinase inhibitor, Y27632, at least one cell culture additive selected from the group consisting of N2 and B27, SB202190, insulin, fibroblast growth factor 10, cholera toxin, ITS cell culture additive, hepatocyte growth factor, 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 with 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 first and the second end of the pipe are connected with each other,

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 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.

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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 to 40. Mu.M, more preferably 2.5 to 20. 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 hepatocyte growth factor is preferably 1.25-20 ng/mL, more preferably 1.25-10 ng/mL;

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

(5) The concentration of SB202190 is preferably 5 to 40nM, more preferably 5 to 20nM;

(6) The concentration of the fibroblast growth factor 10 is preferably 2.5-20 ng/mL, more preferably 2.5-10 ng/mL;

(7) The concentration of cholera toxin is preferably 1.25-20 ng/mL, more preferably 2.5-10 ng/mL;

(8) The concentration of insulin is preferably 2.5 to 40. Mu.g/mL, more preferably 2.5 to 20. Mu.g/mL;

(9) The concentration of Y27632 is preferably 2.5-40. Mu.M, more preferably 5-20. Mu.M;

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

(11) The nonessential amino acid is one or more selected from the group consisting of glycine, alanine, asparagine, aspartic acid, glutamic acid, proline and serine, and the concentration of the nonessential amino acid is preferably 12.5 to 200. Mu.M, more preferably 100 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 liver cancer suspension organoid. In the method for culturing the liver cancer suspension organoid, the liver cancer primary cells are subjected to suspension culture by using the liver cancer suspension organoid culture medium. "suspension culture" refers to a tissue culture system for culturing single cells and small cell clusters in a liquid medium or a low-adsorption culture dish under constant stirring or shaking, and is a culture mode of anchorage-independent cells.

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

1. Separating a sample from a liver cancer solid tumor tissue to obtain a liver cancer primary cell. The treatment process comprises the following steps:

(1) Separating a liver cancer tissue sample, 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 a 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, supernatant is discarded after centrifugation, 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 1640 culture medium, collagenase II (1-2 mg/mL), collagenase IV (1-2 mg/mL), DNA enzyme (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 liver cancer suspension organoid culture medium, and culturing the liver cancer primary cells obtained in the step.

Resuspending and counting the liver cancer primary cells obtained in the step 1 by using the liver cancer suspension organoid culture medium of the invention, and carrying out cell counting according to the proportion of 1-5 multiplied by 10 ⁵ The density of each hole is inoculated on a low adsorption culture plate, and liver cancer suspension organoid culture medium with corresponding volume is added for amplification culture.

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

(1) Culturing the liver cancer organoid by using the culture method of the liver cancer suspension organoid;

(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 liver cancer organoid culture is improved and reaches over 90 percent;

(2) Ensuring that the in vitro primary cultured liver cancer organs can keep the pathological characteristics of patients;

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

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

(5) The culture method does not need matrigel, has simple and convenient culture and passage operation, and is suitable for large-scale culture;

(6) The liver 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-1K are graphs showing the effect of different concentrations of factors added to liver cancer suspension organoid media of the present invention on proliferation of liver cancer organoids.

FIGS. 2A-2D are photographs of observing a liver cancer suspension organoid cultured using the liver cancer suspension organoid medium of the present invention using a microscope, wherein FIG. 2A shows a photograph of an organoid cultured from a sample GL-003 after 7 days; FIG. 2B shows a photograph of organoids obtained from culture of sample GL-006 after 7 days; FIG. 2C shows a photograph of organoids obtained from culture of sample GL-008 after 7 days; FIG. 2D shows photographs of organoids obtained from specimen GL-013 culture after 7 days.

FIG. 3A is a diagram showing pathological and immunohistochemical identification of liver cancer suspension organoids cultured using the liver cancer suspension organoid culture medium of the present invention on sample GL-006; FIG. 3B is a view showing pathological and immunohistochemical identification of the specimen GL-006 primary tissue.

FIGS. 4A-4C are graphs showing the results of culturing a liver cancer organoid using the liver cancer suspension organoid culture medium of the present invention and a conventional culture medium, wherein FIG. 4A shows photographs of liver cancer suspension organoids obtained after 7 days of culture using the HC-XF culture medium of the present invention; FIG. 4B shows photographs of liver cancer suspended organoids obtained after 7 days of culture with Laura's medium; FIG. 4C shows a histogram comparing the relative sizes of HC suspension organoids cultured in HC-XF medium and Laura medium.

FIGS. 5A and 5B show the results of different drug susceptibility tests on liver cancer organoids cultured using the liver cancer suspension organoid culture medium of the present invention, wherein FIG. 5A shows photographs of organoids grown after 5 days of drug treatment with different concentrations of test drugs; FIG. 5B shows a bar graph of the inhibition rate of test drugs at different concentrations for inhibiting growth of hepatoma organoids.

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 and are not intended to limit 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) benzene Sulfonamide 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 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 (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): into 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 analogously to compound 1 and the structure and mass spectral data are shown in the table below.

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Example 1 Effect of factors added to the culture Medium for liver cancer suspension organoids on proliferation of liver cancer suspension organoids

(1) Preparation of liver cancer suspension 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 liver cancer suspension organoid culture medium containing different additive components.

(2) Isolation and processing of primary liver cancer cells

1 sample selection

The tissue sample of the liver cancer solid tumor (during operation) is obtained from patients by professional medical staff of professional medical institutions, and the patients sign informed consent. Intraoperative sample 0.25cm ³ And the commercial tissue preservation solution (manufacturer: miltenyi Biotec) is adopted for storage and transportation.

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 was removed from the 4 ℃ freezer 30 minutes earlier and tissue digest 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 of basal 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 discarding the supernatant, observing whether there are blood cells, if there are blood cells, adding 8mL of blood cell lysate (purchased from Sigma), mixing, lysing for 20 minutes at 4 ℃, reversing and mixing once, and centrifuging at 1500rpm at 25 ℃ for 4 minutes;

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

4 cell count and treatment

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 liver cancer suspension organoids

Resuspending and counting the liver cancer primary cells obtained in the step by using precooled DMEM/F12, and carrying out cell counting according to the proportion of 1-5 multiplied by 10 ⁵ The cells were inoculated in a 24-well low-adsorption plate at a density, and then the medium shown in Table 1 which had been previously returned to room temperature was added thereto, and the medium was changed every three days to carry out scale-up culture. And 7 days later, photographing the cultured organoids, measuring and counting the diameters of the organoids, and comparing the promotion effect of each factor on the proliferation of the liver cancer suspension organoids. 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	Epidermal growth factor EGF	R&D	5ng/mL	○
					3	R-spondin1	R&D	20ng/mL	○
4	Prostaglandin E2	Tocris	0.5μM	○
					5	Insulin	Peprotech	10μg/mL	+
6	B27	Gibco	1:50	+
					7	A8301	MCE	100nM	-
8	SB202190	MCE	10nM	+
					9	Basic fibroblast growth factor bFGF	R&D	10ng/mL	-
10	Hydrocortisone	Sigma	10ng/mL	○
					11	Noggin	R&D	30ng/mL	○
12	Fetal bovine serum FBS	Excell		5％						+
					13	Insulin-like growth factor-1 IGF-1	R&D	45ng/mL	○
14	Keratinocyte growth factor KGF	R&D	5ng/mL	○
					15	GlutaMAX	Gibco	1:100	+
16	Non-essential amino acids	Corning	100μM	+
					17	Dexamethasone	MCE	0.1μM	○
18	Neuregulin 1NRG1	sino biological	5ng/mL	○
					19	Y27632	MCE	10μM	+
20	ITS cell culture additive	Gibco	1:100	+
					21	Compound 1	Preparation examples	5μM	+
22	CHIR99021	MCE	2.5μM	-
					23	Hepatocyte growth factor HGF	R&D	5ng/mL	+
24	Cholera toxin CTX	MCE	5ng/mL	+
					25	Fibroblast growth factor 10	R&D	5ng/mL	+

Wherein "+" indicates that the culture medium added with the additive has the function of promoting proliferation of at least two cases of liver cancer suspended organoids separated from liver cancer tissues compared with a basic culture medium; "-" indicates that the medium added with the additive shows the function of inhibiting proliferation on at least one example of liver cancer suspended organoids separated from liver 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 liver cancer suspension organoids isolated from liver cancer tissue.

From the above results, factors such as B27, hepatocyte Growth Factor (HGF), ITS cell culture supplement, Y27632, SB202190, fibroblast growth factor 10 (FGF 10), insulin, cholera Toxin (CTX), glutaMAX, compound 1, non-essential amino acids, and the like were selected and subjected to further culture experiments.

Example 2 proliferation of liver cancer suspension organoids by Medium supplements at different concentrations

Liver cancer primary cells were obtained from intraoperative tissue samples (No. GL-003, GL-004) according to the method of (2) in example 1, and organoid culture was performed using the medium formulation in Table 2 below.

TABLE 2 culture Medium formulation (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 a suspension organoid on the basis of formula 1, and the final concentrations of B27 are 1; and control wells (BC) were set using medium of formula 1. The final concentration of other additive factors in the series of media was the same as HC-XF media. The following experiments for formulations 1 to 11 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 HGF is added into a 96-well plate inoculated with a suspension organoid on the basis of the formula 2, and the final concentrations of HGF are 1.25ng/mL, 2.5ng/mL, 5ng/mL, 10ng/mL and 20ng/mL respectively; and control wells (BC) were set using medium of formula 2.

When the culture medium of formula 3 is used, 200 μ L of prepared ITS cell culture additive per well is added to a 96-well plate inoculated with organoids on the basis of formula 3, and the final concentrations of ITS cell culture additive are 1; and control wells (BC) were set using medium of formula 3.

When the culture medium of formula 4 is used, 200 μ L of prepared Y27632 per well is added to a 96-well plate inoculated with organoids on the basis of formula 4, and the final concentrations of Y27632 are 2.5 μ M, 5 μ M, 10 μ M, 20 μ M and 40 μ M, respectively; and control wells (BC) were set using medium of formula 4.

When the medium of formula 5 was used, 200. Mu.L of prepared SB202190 was added to each well of formula 5 in a 96-well plate inoculated with organoids, respectively, at final concentrations of 2.5nM, 5nM, 10nM, 20nM, and 40nM SB 202190; and control wells (BC) were set using medium of formulation 5.

When the culture medium of the formula 6 is used, 200 mu L of prepared FGF10 is added into a 96-well plate inoculated with organoids on the basis of the formula 6, wherein the final concentration of the FGF10 is respectively 2.5ng/mL, 5ng/mL, 10ng/mL, 20ng/mL and 40ng/mL; and control wells (BC) were set using medium of formula 6.

When the culture medium of the formula 7 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 7, wherein the final concentration of the insulin is respectively 2.5 mu g/mL, 5 mu g/mL, 10 mu g/mL, 20 mu g/mL and 40 mu g/mL; and control wells (BC) were set using medium of formulation 7.

When the culture medium of the formula 8 is used, 200 mu L of prepared CTX is added into a 96-well plate inoculated with an organoid on the basis of the formula 8, and the final concentration of the CTX is 1.25ng/mL, 2.5ng/mL, 5ng/mL, 10ng/mL and 20ng/mL respectively; and control wells (BC) were set using the medium of formulation 8.

When the medium of formula 9 is used, 200 μ L of prepared GlutaMAX is added to each well of formula 9 in 96-well plates inoculated with organoids, wherein the final concentration of GlutaMAX is 1; and control wells (BC) were set using medium of formulation 9.

When the medium of the formula 10 was used, 200. Mu.L of the prepared compound 1 was added to each well of the formula 10 in 96-well organoid-inoculated plates, respectively, at final concentrations of 2.5. Mu.M, 5. Mu.M, 10. Mu.M, 20. Mu.M, and 40. Mu.M of the compound 1; and control wells (BC) were set using medium of formula 10.

When the culture medium of formula 11 was used, 200. Mu.L of the prepared nonessential amino acids per well were added to each 96-well plate inoculated with organoids based on formula 11, and the final concentrations of the nonessential amino acids were 12.5. Mu.M, 25. Mu.M, 50. Mu.M, 100. Mu.M, and 200. Mu.M, respectively; and control wells (BC) were set using medium of formula 11.

After 14 days, the cultured organoids were photographed, and the sizes of diameters of the organoids were measured and counted, and the effects of the concentrations of the factors on the proliferation of the liver cancer organoids were compared. The data collected for 2 samples are summarized in FIGS. 1A-1K. In FIGS. 1A-1K, the ratio is the ratio of the organoid diameter obtained by 14 days of culture using each medium to the organoid diameter obtained by 14 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 1K, the volume concentration of B27 is preferably 1; the content of the hepatocyte growth factor is preferably 1.25-20 ng/mL, more preferably 1.25-10 ng/mL; the volume concentration of the ITS cell culture additive is preferably 1; the content of Y27632 is preferably 2.5-40 μ M, more preferably 5-20 μ M; the content of SB202190 is preferably 5 to 40nM, more preferably 5 to 20nM; the content of the fibroblast growth factor 10 is preferably 2.5-20 ng/mL, and more preferably 2.5-10 ng/mL; the content of insulin is preferably 2.5 to 40 mug/mL, more preferably 2.5 to 20 mug/mL; the content of cholera toxin is preferably 1.25-20 ng/mL, and more preferably 2.5-10 ng/mL; the volume concentration of GlutaMAX is preferably 1; the content of the MST1/2 kinase inhibitor compound 1 is preferably 2.5 to 40 μ M, more preferably 2.5 to 20 μ M; the content of the non-essential amino acid is preferably 12.5 to 200. Mu.M, more preferably 100 to 200. Mu.M.

Example 3 liver cancer suspension organoid culture and characterization

The primary liver cancer cells (GL-003, GL-006, GL-008, GL-013) obtained by the method described in (2) of example 1 were resuspended and counted in the liver cancer suspension organoid medium HC-XF of the present invention, and the cells were then cultured at 5X 10 ⁵ The culture medium is inoculated in a 24-hole low adsorption culture plate in a density, 1mL of liver cancer suspension organoid culture medium is added, and the culture medium is replaced once every three days for amplification culture.

On day 7, the cultured liver cancer-like suspended organs were observed using a microscope (EVOS M500, invitrogen). FIGS. 2A-2D are photographs of suspended liver cancer organoids taken under 10-fold objective lens after culturing samples GL-003, GL-006, GL-008, GL-013 for 7 days. The liver cancer suspension organoids are regular spherical under the microscope, and the surface is smooth.

And performing pathological and immunohistochemical identification on the cultured liver cancer suspended organoids, and performing pathological and immunohistochemical identification on corresponding tissue samples at the same time, and comparing the results of the organoids and the original tissues for consistency.

FIG. 3A is the result of pathological and immunohistochemical identification of suspended liver cancer organoids obtained from GL-006 cultured in vitro, which are pictures taken under 20-fold objective lens. As shown in fig. 3A, HE results show that organoid structural morphology is cancer tissue morphology; CK19, heppar-1 and Ki67 expression, which indicates that the sample is liver cancer. FIG. 3B is a graph showing the pathological and immunohistochemical results of GL-006 corresponding tissues before culture. The result shows that the diagnosis result of the liver cancer organoid cultured by the culture medium HC-XF of the invention is consistent with the diagnosis result of the liver cancer tissue before culture.

Example 4 comparison of culture Effect with existing organoid Medium

(1) Preparation of control Medium

The medium used in the formulation literature (Laura et al, nat Med.2017,23 (12): 1424-1435) was formulated as Advanced DMEM/F12 medium (from Corning Corp.) +1. Hereinafter referred to as "Laura" medium.

(2) Liver cancer suspension organoid culture

Liver cancer primary cells were obtained from intraoperative tissue sample GL-018 according to the method of (2) of example 1, and suspension organoid culture was carried out according to the method of example 3 using HC-XF medium and Laura medium, respectively.

On the 7 th day of culture, the cultured liver cancer suspended organoids were observed using a microscope (EVOS M500, invitrogen). FIGS. 4A and 4B are photographs taken under 4-fold objective lens of organoids cultured with HC-XF medium and Laura medium, respectively, and FIG. 4C is a bar graph comparing the relative sizes of organoids cultured with the two media.

As shown in FIGS. 4A-4C, HC-XF medium significantly promoted the expansion and culture of liver cancer organoids compared to Laura medium.

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

(1) Suspension organoid culture of liver cancer

Primary liver cancer cells were isolated from a liver cancer intraoperative sample (GL-006) by the method of (2) in example 1, and suspension organoid culture was performed using HC-XF medium, and drug screening was performed when the diameter of liver cancer organoid suspension exceeded 50 μm.

(2) Screening drug formulations

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

TABLE 3 preparation of epirubicin, doxorubicin and daunorubicin additive solution

Epirubicin	Concentration of formulation (mM)	2	0.2
				Doxorubicin	Concentration of formulation (mM)	2	0.2
Daunorubicin	Concentration of formulation (mM)	2	0.2

(3) Dosing

The prepared medicine is taken out and placed at room temperature, and the medicine is diluted 1000 times by HC-XF culture medium for standby. The organoids obtained by culturing according to step (1) were removed from the incubator, transferred to a 15ml centrifuge tube, and centrifuged at 1500rpm for 5 minutes. Resuspending organoid with HC-XF culture medium, counting, adjusting organoid concentration to 50/50 μ L, adding 50 μ L of the adjusted organoid suspension into 96-well plate, adding 50 μ L of diluted medicine, sterilizing the surface of 96-well plate, transferring to incubator, culturing, and measuring organoid activity after 5 days.

(4) Organoid viability test

The CellTiter-Glo luminescent reagent (purchased from Promega) is taken out from a refrigerator at 4 ℃,10 ml of the reagent is put into a sample adding groove, a 96-well plate to be detected is taken out from an incubator, 20 mu L of the CellTiter-Glo luminescent reagent is added into each well, the mixture is evenly mixed after standing for 10 minutes, and the detection is carried out by using a multifunctional microplate reader (Envision of Perkin Elmer).

(5) Data processing

The chemical luminescence value of the culture well on the fifth day is determined according to the formula, wherein the drug inhibition ratio (%) =100% - (day five) _{Drug treatment group} Day zero culture well chemiluminescence values _{Drug treatment group} ) /(day five 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 taken with a 4-fold objective microscope (EVOS M500, invitrogen) showing the organoid growth after 5 days of drug addition treatment, and FIG. 5B is a photograph showing the inhibition rate of the test drug at different concentrations for inhibiting the growth of the liver cancer organoidA histogram.

As can be confirmed from FIG. 5A, organoids cultured using the liver cancer suspension organoid medium of the present invention were significantly inhibited from growing after high concentration drug treatment. FIG. 5B is a bar graph of the inhibition rate of three test drugs at different concentrations for inhibiting growth of hepatoma organoids. 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 anti-tumor drugs, the anti-tumor drugs have stronger effect of inhibiting the growth of the organoid under high concentration, and have larger difference in the effect of inhibiting the growth of the organoid under low concentration, which shows that the organoids of the same patient have different effectiveness and sensitivity to different drugs. According to the result, the effectiveness and the effective dosage of the medicine can be judged when the liver cancer patient clinically uses the medicine.

Industrial applicability

The invention provides a culture medium and a culture method for liver cancer suspension organoid culture, which can be used for evaluating and screening the curative effect of a medicament by using the cultured organoids. 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 (11)

1. A culture medium for suspension organoids of liver cancer comprising an MST1/2 kinase inhibitor, Y27632, at least one cell culture additive selected from the group consisting of N2 and B27, SB202190, insulin, fibroblast growth factor 10, cholera toxin, ITS cell culture additives, hepatocyte growth factor, 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 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 the group consisting of 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,

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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 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:

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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 2.5-40 mu M;

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

the concentration of the hepatocyte growth factor is 1.25-20 ng/mL;

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

the concentration of the Y27632 is 2.5-40 mu M;

the concentration of the SB202190 is 5-40 nM;

the concentration of the fibroblast growth factor 10 is 2.5-20 ng/mL;

the concentration of the insulin is 2.5-40 mug/mL;

the concentration of the cholera toxin is 1.25-20 ng/mL;

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 12.5-200 mu M.

7. 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 2.5-20 mu M;

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

the concentration of the hepatocyte growth factor is 1.25-10 ng/mL;

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

the concentration of the Y27632 is 5-20 mu M;

the concentration of the SB202190 is 5-20 nM;

the concentration of the fibroblast growth factor 10 is 2.5-10 ng/mL;

the concentration of the insulin is 2.5-20 mug/mL;

the concentration of the cholera toxin is 2.5-10 ng/mL;

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 100-200 mu M.

8. 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.

9. The culture medium according to any one of claims 1 to 5, wherein the culture medium is free of Wnt agonists, R-spondin family proteins, noggin proteins, or BMP inhibitors.

10. A culture method of liver cancer suspension organoids is characterized by comprising the following steps:

(1) Separating a sample from a liver cancer solid tumor tissue to obtain a liver cancer primary cell;

(2) Preparing a culture medium of the liver cancer suspension organoid according to any one of claims 1-9, and performing suspension culture on the liver cancer primary cells obtained in step (1).

11. A method for evaluating or screening a drug for treating liver cancer, comprising the steps of:

(1) Culturing a liver cancer suspension organoid using the method of culturing a liver cancer suspension organoid according to claim 10;

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

(3) Adding the diluted medicine to the suspended organoids obtained by culturing in (1);

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

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