Patent Application
20250129329
The invention relates to the field of biotechnology, and in particular to a culture medium for hepatoma organoid culture, a method for culturing hepatoma organoid using the culture medium, and the application thereof in the efficacy evaluating and screening of drugs.
In recent years, postoperative adjuvant chemotherapy for liver cancer, including postoperative TACE treatment, oral medication treatment, etc., has gradually gained the attention and recognition of clinical doctors as a new type of adjuvant treatment. However, due to the lack of standardized chemotherapy regimens, conventional chemotherapies based on experiences may ignore individual differences and lead to blindness, and thus, the efficacy has always been unsatisfied, with the efficacy rate of less than 20% in monotherapy or combination therapy (Jindal A, Thadi A, Shailubhai K. Hepatocellular Carcinoma: Etiology and Current and Future Drugs [J]. J Clin Exp Hepatol, 2019, 9 (2): 221-232). Although emerging targeted drugs have reduced toxic side-effects to a certain extent, the quantity of drugs is too small, the treatment cost is expensive, and the effectiveness varies among individuals, making it difficult to meet the treatment needs of most patients. Due to the lack of an effective liver cancer drug sensitivity testing system, precise chemotherapy cannot be achieved. Therefore, matching the in vitro drug sensitivity results of liver cancer with clinical in vivo reactions becomes the key to treatment.
Traditional clinical drug sensitivity testing mostly uses two-dimensional cell culture. However, two-dimensional cultured cells can only simulate the physiological conditions of tissue to a limited extent, without real tissue structure in vivo, which may prone to low differentiation levels and loss of cell physiological functions, making it difficult to predict actual clinical results from the obtained experimental results. Organoids belong to three-dimensional (3D) cell cultures, which are mainly derived from human embryonic stem cells, induced pluripotent stem cells, and adult stem cells with differentiation ability. Endogenous tissue stem cells, which play an important role in maintaining the functional morphology of each organ, exist in different tissues and organs. These stem cells can self organize and form a mini structure with a diameter of only a few millimeters under certain induction conditions in vitro. Tumor organoids are miniature 3D tumor cell models cultured in the laboratory from primary tumors taken from patient's body. Tumor organoids can highly simulate the characteristics of the source tumor tissue, preserving tumor heterogeneity among individuals, and can be used for functional testing, such as high-throughput drug screening and personalized precision therapy.
Currently, culture methods for hepatoma organoid often use basic culture media (DMEM or DMEM/F12), R-spindin-1, Noggin, and some expensive protein factors, resulting in higher costs for organoid culture; and the complex operation and the difficulty of this technology have limited its large-scale commercial applications. Therefore, it is necessary to develop a low-cost, simple, and of high success rate culture method and culture medium for organoid.
In order to solve the aforementioned technical problems, the invention provides a culture medium and culture method for rapidly expanding hepatoma organoid in vitro.
One aspect of the invention is to provide a culture medium for hepatoma organoid, comprising an MST1/2 kinase inhibitor, at least one cell culture additive selected from N2 and B27, a hepatocyte growth factor, an ITS cell culture additive, Y27632, dexamethasone, Neuregulin-1, insulin, an epidermal cell 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 a solvate thereof,
In a preferable embodiment, the MST1/2 kinase inhibitor comprises a compound of Formula (Ia) or a pharmaceutically acceptable salt, or a solvate thereof,
Preferably, the MST1/2 kinase inhibitor is at least one selected from the following compounds or a pharmaceutically acceptable salt, or a solvate thereof.
Most preferably, the MST1/2 kinase inhibitor of the invention is Compound 1.
In the embodiment of the invention, the amounts of the components in the culture medium of the invention satisfies any one or more or all of the following conditions:
In the embodiment of the invention, the culture medium further comprises an initial medium selected from the group consisting of DMEM/F12, DMEM, F12 or RPMI-1640; and one or more antibiotics selected from the group consisting of streptomycin/penicillin, amphotericin B and Primocin.
In a preferred embodiment, in case of using streptomycin/penicillin as the antibiotics, the concentration range of streptomycin is 25-400 μg/mL, the concentration range of penicillin is 25-400 U/mL; in case of using amphotericin B as the antibiotics, the concentration range is 0.25-4 μg/mL; and in case of using Primocin as the antibiotics, the concentration range is 25-400 μg/mL.
The invention also provides a method for culturing hepatoma organoid. In the method for culturing hepatoma organoid of the invention, the hepatoma organoid is cultured using the culture medium for hepatoma organoid of the invention.
The method for culturing hepatoma organoid of the invention comprises the following steps.
Wherein, the formulation of the basic medium comprises an initial medium selected from the group consisting of DMEM/F12, DMEM, F12 or RPMI-1640; and one or more antibiotics selected from the group consisting of streptomycin/penicillin, amphotericin B and Primocin. The formulation of the tissue digestion solution comprises 1640 medium, collagenase II (1-2 mg/mL), collagenase IV (1-2 mg/mL), DNase (50-100 U/mL), hyaluronidase (0.5-1 mg/mL), calcium chloride (1-5 mM), bovine serum albumin BSA (5-10 mg/mL).
The primary liver cancer cells obtained in above step 1 are resuspended with the culture medium for hepatoma organoid of the invention and counted. The cell density is diluted to 5-10×106 cells/mL, and the diluted cell suspension is added with an equal volume of Matrigel matrix gel and mixed well. Then, the mixture is inoculated onto a multi well plate and the plate is placed in a culture incubator for 30-60 minutes until Matrigel is completely coagulated. Then, the culture medium for hepatoma organoid is added for expansion and cultivation.
The invention also provides a method for evaluating or screening a drug for treating liver cancer, comprising the following steps:
The beneficial effects of the invention include:
In order to better understand the invention, it is further described below in combination with the embodiments and the drawings. The following examples are provided only for the purpose of illustrating, but not for limiting the invention.
In the specification, MST1/2 kinase inhibitor refers to any inhibitor that directly or indirectly negatively regulates MST1/2 signaling. Generally, MST1/2 kinase inhibitors reduce the activity of MST1/2 kinase by, for example, binding to the same. Since MST1 and MST2 have similar structures, MST1/2 kinase inhibitors may be, for example, compounds that bind to MST1 or MST2 and reduce the activity thereof.
Methyl 2-amino-2-(2,6-difluorophenyl)acetate (A2): 2-amino-2-(2,6-difluorophenyl) acetic acid (2.0 g) and then methanol (30 ml) were added into a round bottom flask, followed by addition of thionyl chloride (1.2 ml) dropwise under an ice bath. The reaction system was reacted overnight at 85° C. After the completion of the reaction, the system was evaporated under reduced pressure to dry the solvent, and the obtained white solid was directly used in the next step.
Methyl 2-((2-chloro-5-nitropyrimidin-4-yl)amino)-2-(2,6-difluorophenyl) acetate (A3): methyl 2-amino-2-(2,6-difluorophenyl)acetate (2 g) and then acetone (30 ml) and potassium carbonate (2.2 g) were added into a round bottom flask, and then the system was cooled to −10° C. with an ice salt bath, and then a solution of 2,4-dichloro-5-nitropyrimidine (3.1 g) in acetone was slowly added. The reaction system was stirred overnight at room temperature. After the completion of the reaction, the reaction mixture was filtered, the solvent was removed from the filtrate under reduced pressure, and the residue was purified by pressurized silica gel column chromatography to obtain compound A3. LC/MS: M+H 359.0.
2-Chloro-7-(2,6-difluorophenyl)-7,8-dihydropteridin-6 (5H)-one (A4): methyl 2-((2-chloro-5-nitropyrimidin-4-yl)amino)-2-(2,6-difluorophenyl)acetate (2.5 g) and then acetic acid (50 ml) and iron powder (3.9 g) were added into a round bottom flask. The reaction system was stirred at 60° C. for two hours. After the completion of the reaction, the reaction system was evaporated under reduced pressure to dry the solvent, and the resultant was neutralized to alkaline with saturated sodium bicarbonate solution and was extracted with ethyl acetate. The organic phase was washed with water and saturated brine and dried with anhydrous sodium sulfate. The organic phase was filtered and evaporated to dryness under reduced pressure to obtain a crude product. The crude product was washed with diethyl ether to obtain compound A4. LC/MS: M+H 297.0.
2-Chloro-7-(2,6-difluorophenyl)-5,8-dimethyl-7,8-dihydropteridin-6 (5H)-one (A5): 2-chloro-7-(2,6-difluorophenyl)-7,8-dihydropteridin-6 (5H)-one (2 g) and N,N-dimethylacetamide (10 ml) were added into a round bottom flask, and cooled to −35° C., followed by addition of iodomethane (0.9 ml) and then sodium hydride (615 mg), and the reaction system was stirred for two hours. After the completion of the reaction, the reaction mixture was quenched with water, and extracted with ethyl acetate. The organic phase was washed with water and saturated brine, respectively, and dried with anhydrous sodium sulfate. The organic phase was filtered and evaporated to dryness under reduced pressure to obtain a crude product. The crude product was washed with diethyl ether to obtain compound A5. LC/MS: M+H 325.0.
4-((7-(2,6-Difluorophenyl)-5,8-dimethyl-6-oxo-5,6,7,8-tetrahydropteridin-2-yl)amino)benzsulfamide (1): 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) were added into a round bottom flask. The reaction system was stirred at 120° C. overnight. After the completion of the reaction, the reaction mixture was filtered, and washed with methanol and diethyl ether to obtain compound 1. LC/MS: M+H 461.1.
Other MST1/2 inhibitor compounds of the invention were synthesized via the method similar to that of Compound 1, and their structures and mass spectrum data are shown in the following table.
First, a basic medium containing initial medium was prepared. The initial medium can be selected from commonly used DMEM/F12, DMEM, F12, or RPMI-1640. In this embodiment, the formulation of the basic medium is: DMEM/F12 medium (purchased from Corning)+100 μg/mL Primocin (purchased from InvivoGen, 0.2% (v/v), commercial product has a concentration of 50 mg/ml).
Different types of additives (see Table 1) were added to the basic medium to prepare culture mediums for hepatoma organoid with different components.
Tissue samples (intraoperative) of liver cancer solid tumor were obtained from patients by professional medical practitioner of professional medical institutions, and all patients have signed the informed consent. Intraoperative samples having the size of 0.25 cm3 were stored and transported using commercial tissue preservation solution (manufacturer: Miltenyi Biotec).
After subjecting to surface sterilization, 15 mL sterile centrifuge tubes, pipettors, 10 mL pipettes, sterile pipette tips, etc., were put in an ultra-clean workbench for ultraviolet irradiation of 30 minutes. The basic medium was taken out from a 4° C. refrigerator 30 minutes in advance, and the tissue digestion solution was taken out from a −20° C. refrigerator 30 minutes in advance.
Tissue digestion solution: 1640 medium (Corning, 10-040-CVR), collagenase II (2 mg/mL), collagenase IV (2 mg/mL), DNase (50 U/mL), hyaluronidase (0.75 mg/mL), calcium chloride (3.3 mM), BSA (10 mg/mL).
Collagenase II, collagenase IV, DNase, and hyaluronidase mentioned above were all purchased from Sigma Corporation; calcium chloride was purchased from Sangon Biotech (Shanghai) Co., Ltd.; BSA was purchased from Biofroxx Corporation.
The primary liver cancer cells obtained in the above steps were resuspended with pre-cooled DMEM/F12 and counted. The cell density was diluted to 5-10×106 cells/mL and 400 μL of the diluted cell suspension was added with an equal volume of Matrigel matrix gel (Corning) and mixed gently and well. Then, the mixture was inoculated at 5 μL/well onto a 96-well plate. The inoculated plate was placed into an incubator for 30 minutes until Matrigel is completely coagulated. Then, the culture mediums as shown in Table 1 that have been come to room temperature in advance were added, respectively, and the culture mediums were renewed every three days for expansion and cultivation. After 7 days, the cultured organoids were photographed and the diameters of the organoids were measured and compiled, so as to compare the promoting effects of various factors on the proliferation of hepatoma organoids. As the experimental control, the basic medium without any additives was used. The experimental results are shown in Table 1.
Wherein, “+” indicates that compared with the Basic Medium, the medium added with the additive(s) has the effect of promoting the proliferation of hepatoma organoids isolated from liver cancer tissue in at least two cases; “−” indicates that the medium added with the additive(s) has the effect of inhibiting the proliferation of hepatoma organoids isolated from liver cancer tissue in at least one case; “∘” indicates that the medium added with the additive(s) has no significant effect on the proliferation of hepatoma organoids isolated from liver cancer tissue in at least two cases.
According to the above results, factors including B27, hepatocyte growth factor (HGF), ITS cell culture additive, Y27632, dexamethasone, Neuregulin-1 (NRG1), insulin, epidermal growth factor (EGF), GlutaMAX, Compound 1, and non-essential amino acids were selected for further experiments of culture.
Primary liver cancer cells were obtained from intraoperative tissue samples (Nos. GL-003, GL-004) in accordance with the method described in Section (2), Example 1, and the organoids were cultured using the culture medium formulations as shown in Table 2 below.
When using the culture medium of Formulation 1, to a 96-well plate inoculated with organoids was added the prepared B27 in addition to the medium of Formulation 1, with 200 μL per well, such that the final concentrations of B27 were 1:25, 1:50 and 1:100, respectively; a well of Blank Control (BC) was set using the medium of Formulation 1. The final concentrations of other factors added in this series of culture mediums were the same as those in HC-3 culture medium. The experiments of Formulations 1-11 below were also conducted in the same way and will not be described in details.
When using the culture medium of Formulation 2, to a 96-well plate inoculated with organoids was added the prepared HGF in addition to the medium of Formulation 2, with 200 μL per well, such that the final concentrations of HGF were 1 ng/ml, 5 ng/mL and 25 ng/mL, respectively; a well of Blank Control (BC) was set using the medium of Formulation 2.
When using the culture medium of Formulation 3, to a 96-well plate inoculated with organoids was added the prepared ITS cell culture additive in addition to the medium of Formulation 3, with 200 μL per well, such that the final concentrations of ITS cell culture additive were 1:300, 1:100 and 1:30, respectively; a well of Blank Control (BC) was set using the medium of Formulation 3.
When using the culture medium of Formulation 4, to a 96-well plate inoculated with organoids was added the prepared Y27632 in addition to the medium of Formulation 4, with 200 μL per well, such that the final concentrations of Y27632 were 3 μM, 10 μM and 30 μM, respectively; a well of Blank Control (BC) was set using the medium of Formulation 4.
When using the culture medium of Formulation 5, to a 96-well plate inoculated with organoids was added the prepared dexamethasone in addition to the medium of Formulation 5, with 200 μL per well, such that the final concentrations of dexamethasone were 0.01 μM, 0.1 μM and 1 μM, respectively; a well of Blank Control (BC) was set using the medium of Formulation 5.
When using the culture medium of Formulation 6, to a 96-well plate inoculated with organoids was added the prepared NRG1 in addition to the medium of Formulation 6, with 200 μL per well, such that the final concentrations of NRG1 were 1 ng/ml, 5 ng/ml and 25 ng/ml, respectively; a well of Blank Control (BC) was set using the medium of Formulation 6.
When using the culture medium of Formulation 7, to a 96-well plate inoculated with organoids was added the prepared insulin in addition to the medium of Formulation 7, with 200 UL per well, such that the final concentrations of insulin were 1 μg/mL, 3 μg/mL and 10 μg/mL, respectively; a well of Blank Control (BC) was set using the medium of Formulation 7.
When using the culture medium of Formulation 8, to a 96-well plate inoculated with organoids was added the prepared EGF in addition to the medium of Formulation 8, with 200 μL per well, such that the final concentrations of EGF were 2 ng/ml, 6 ng/ml and 18 ng/ml, respectively; a well of Blank Control (BC) was set using the medium of Formulation 8.
When using the culture medium of Formulation 9, to a 96-well plate inoculated with organoids was added the prepared GlutaMAX in addition to the medium of Formulation 9, with 200 μL per well, such that the final concentrations of GlutaMAX were 1:300, 1:100 and 1:30, respectively; a well of Blank Control (BC) was set using the medium of Formulation 9.
When using the culture medium of Formulation 10, to a 96-well plate inoculated with organoids was added the prepared Compound 1 in addition to the medium of Formulation 10, with 200 μL per well, such that the final concentrations of Compound 1 were 2.5 M, 5 μM and 10 μM, respectively; a well of Blank Control (BC) was set using the medium of Formulation 10.
When using the culture medium of Formulation 11, to a 96-well plate inoculated with organoids was added the prepared non-essential amino acids in addition to the medium of Formulation 11, with 200 μL per well, such that the final concentrations of non-essential amino acids were 50 μM, 100 μM and 200 μM, respectively; a well of Blank Control (BC) was set using the medium of Formulation 11.
After 10 days, the cultured organoids were photographed and the diameters of the organoids were measured and compiled, so as to compare the promoting effects of various factor concentrations on the proliferation of hepatoma organoids. The data collected from two samples were summarized and shown in
According to the results of
The primary liver cancer cells (GL-003, GL-006, GL-008, GL-013) obtained by the method described in Section (2), Example 1 were resuspended using the HC-3 culture medium for hepatoma organoid of the invention and counted. The cell density was diluted to 5-10×106 cells/mL and 400 μL of the diluted cell suspension was added with an equal volume of Matrigel matrix gel (Corning) and mixed gently and well. Then, the mixture was inoculated at 50 μL/well onto a 24-well plate. The inoculated plate was placed into an incubator for 30 minutes until Matrigel is completely coagulated. Then, the HC-3 culture medium for hepatoma organoid that has been come to room temperature in advance were added, with 500 μL per well, and the culture medium were renewed every three days for expansion and cultivation.
On Day 10-15, the cultured hepatoma organoids were observed using a microscope (Invitrogen, EVOS M500).
Pathological and immunohistochemical identification was performed on the cultured hepatoma organoids, and the corresponding tissue samples were also sent for pathological and immunohistochemical identification to compare the consistency of results between the organoids and tissues.
Culture medium used in a literature (Laura et al., Nat Med. 2017, 23 (12): 1424-1435) was prepared, which has the formulation of: Advanced DMEM/F12 medium (purchased from Corning)+1:100 Penicillin/Streptomycin (purchased from Corning)+1:100 GlutaMAX (purchased from Corning)+10 mM HEPES (purchased from Thermo Fisher)+1:50 B27 (purchased from Gibco)+1:100 N2 (purchased from Gibco)+1.25 mmol/L N-acetylcysteine (purchased from MCE)+10 mmol/L nicotinamide (purchased from MCE)+10 nM gastrin (purchased from MCE)+50 ng/ml epidermal growth factor (purchased from R&D)+100 ng/ml fibroblast growth factor 10 (purchased from sino biological)+25 ng/ml hepatocyte growth factor (purchased from R&D)+10 μmol/L Forskolin (purchased from MCE)+5 μmol/L A8301 (purchased from MCE)+10 μmol/L Y27632 (purchased from MCE)+3 nmol/L dexamethasone (purchased from MCE). Hereinafter, it referred to as Laura culture medium.
Primary liver cancer cells were obtained from intraoperative tissue sample GL-018 according to the method described in Section (2), Example 1, and were cultured to obtain organoids by using HC-3 culture medium and Laura culture medium, respectively, according to the method described in Example 3.
On Day 25 of cultivation, the cultured hepatoma organoids were observed using a microscope (Invitrogen, EVOS M500).
According to the results of
Primary liver cancer cells were isolated from liver cancer intraoperative sample (GL-006) according to the method described in Section (2), Example 1, and were cultured to obtain organoids by using HC-3 culture medium until the diameters of hepatoma organoids exceeded 50 μm, which were then used for drug screening.
Prepare Three drugs with two concentration gradients were prepared according to the table below (Bortezomib, Aclarubicin, and Doxorubicin; all purchased from MCE) and store for use.
The prepared drugs were taken out and placed at room temperature. The drugs were diluted 1000 times with HC-3 culture medium for later use. The organoids cultured and obtained according to step (1) were taken out from the incubator, the culture mediums were removed from the wells, and the culture mediums containing drugs were slowly added into the wells along the well wall. After the addition of drug, disinfect the surface of the 96-well plate was sterilized and then was transferred to the incubator for further cultivation. After 5 days, the viability of organoids were measured.
CellTiter-Glo luminescent reagent (purchased from Promega) was taken out from a 4° C. refrigerator, and 10 mL of the reagent was added into the loading slot; the 96-well plate for testing was taken out from the incubator, and 20 μL CellTiter-Glo luminescent reagent was added into each well. After standing for 10 minutes followed by mixing well, the test was conducted by using a multi-functional microplate reader (Envision, Perkin Elmer).
According to the formula, Drug inhibition rate (%)=100%−(Chemiluminescence value of drug-treatment well on Day 5/Chemiluminescence value of drug-treatment well on Day 0)/(Chemiluminescence value of DMSO well on Day 5/Chemiluminescence value of DMSO well on Day 0)×100%, the inhibition rates of different drugs were calculated. The results were shown in
From
The invention provides a culture medium and a culture method for hepatoma organoid culture, and the cultured organoids can be used in efficacy evaluating and screening of drugs. Thus, the invention is suitable for industrial applications.
Although the invention has been described in detail herein, the invention is not limited thereto, and those skilled in the art can make modifications according to the principle of the invention. Therefore, any modification made in accordance with the principle of the invention shall be understood as falling within the protection scope of the invention.
| Number | Date | Country | Kind |
|---|---|---|---|
| 202111048845.4 | Sep 2021 | CN | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2021/118657 | 9/16/2021 | WO |
