The invention relates to the field of biomedical technology, and in particular to a culture medium and application thereof, and more specifically to a culture medium for primary ovarian cancer cells, and a culture method and application thereof.
Ovarian cancer refers to a malignant tumor disease that occurs in the ovary, which is one of the common malignant tumors of female reproductive organs, and has an incidence second only to cervical cancer and uterine body cancer. Epithelial ovarian cancer is the most common type of ovarian cancer, followed by malignant ovarian germ cell tumor. Epithelial ovarian cancer has the highest mortality rate among all types of gynecological tumors and may pose a serious threat to women's lives. Ovarian cancer is often asymptomatic in the early stage, but gastrointestinal symptoms such as lower abdominal discomfort, bloating, and decreased appetite may occur in the late stage. The main treatment options for ovarian cancer include surgical resection, drug therapy, and radiotherapy, and the overall prognosis is poor.
Chemotherapy is one of the main treatments for ovarian tumors. Although there are many chemotherapy drugs currently available for clinical use, the clinical treatment effectiveness of ovarian tumors is only about 25%. The main reason is that most chemotherapy drug regimens used by patients are based on the experience of clinicians, undergoing trial-evaluation-medication change and trial-reevaluation without considering the individual differences of patients, not only fails to improve the therapeutic effect of the drugs, but also misses the best treatment period, leading to the tumor entering the advanced stage. In addition, patients are burdened with drug side effects and extremely high medical costs throughout the treatment process.
Therefore, establishing a primary tumor model in vitro and using it to conduct efficient drug screening experiments is a promising solution. At present, the main method for establishing in vitro culture models of primary ovarian tumors is the Patient-Derived tumor Xenograft (PDX) model, which involves transplanting tumor cells from patients into nude mice and examining the therapeutic effects of different anti-tumor drugs. However, the PDX method also has some shortcomings, such as, species differences between humans and mice; long test cycle (more than 4 weeks); high cost (more than 200,000); false positives and false negatives, etc.
In order to solve the aforementioned technical problems, the invention provides a culture medium for primary ovarian cancer cells, and a culture method in vitro and application thereof.
One aspect of the invention is to provide a culture medium for primary ovarian cancer cells, comprising a MST1/2 kinase inhibitor, at least one Rho kinase inhibitor selected from the group consisting of Y27632, fasudil, and H-1152; insulin-transferrin-selenium supplement; insulin; prostaglandin E2; epidermal growth factor (EGF); gastrin; insulin-like growth factor-1; cholera toxin; amphiregulin; N2; and B27.
Wherein the MST1/2 kinase inhibitor comprises a compound of Formula (I) or a pharmaceutically acceptable salt, or a solvate thereof,
wherein,
In a preferable embodiment, the MST1/2 kinase inhibitor comprises a compound of Formula (Ia) or a pharmaceutically acceptable salt, or a solvate thereof,
wherein,
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 an embodiment of the invention, the amount 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.
According to the second aspect, the invention also provides a method for culturing primary ovarian cancer cells in vitro. In the method for culturing primary ovarian cancer cells in vitro of the invention, the primary ovarian cancer cells are cultured in vitro using the culture medium for primary ovarian cancer cells of the invention.
The method for culturing primary ovarian cancer cells in vitro of the invention comprises the following steps:
Wherein, the formulation of the Basal 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 Basal Medium is mixed with matrigel (Corning®, 356231) in a ratio of 25:1 to 100:1 on ice. After plating the cells, the plates are incubated in a 5% CO2 incubator at 37° C. for 25 to 35 minutes, and the supernatant is discarded. The primary ovarian cancer cells obtained in the above step 1 are resuspended with the culture medium for primary ovarian cancer cells of the invention, and the cells are counted. The cell density is adjusted to 2-8×104 cells/cm2. The culture medium for primary ovarian cancer cells of the invention is added, and the cells are cultured in an incubator.
In yet another aspect, the invention also provides a drug screening method for ovarian cancer disease, which includes the following steps:
The technical solution of the invention can achieve the following technical effects:
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 Basal Medium containing initial medium was prepared. The initial medium may be selected from the group consisting of DMEM/F12, DMEM, F12, or RPMI-1640 commonly used in the art. In this example, the formulation of the Basal 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 Basal Medium to prepare culture mediums for primary ovarian cancer cells with different components.
Tissue samples (intraoperative) of ovarian 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, endoscopic samples having the size of 0.025 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 Basal 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 ovarian cancer cells isolated from 2 cases of ovarian cancer tissues (numbered L40 and LQQ) according to the above step (2) were resuspended in the culture medium in Table 1, respectively and the cell number was counted. The Basal Medium was mixed with matrigel (Corning®, 356231) in a 50:1 ratio on ice. 300 μL of the mixture was plated and incubated in a 5% CO2 incubator at 37° C. for 25-35 minutes, preferably 30 minutes. The supernatant was discarded. The culture medium with different components of Table 1 containing primary ovarian cancer cells was added to a 48-well plate with 4×104 cells per well and 500 μL/well. The Basal Medium without addition of any additive was used as a control. After 7-10 days of culture, the cells grew to 85%, and the culture mediums were discarded and the cells were rinsed once with 100 μL 0.05% trypsin (purchased from Gibco) per well. After aspirating the trypsin, 200 μL of 0.05% trypsin was added to each well. The plate was then placed in an incubator at 37° C. and 5% CO2 for reaction of 10 minutes, and the cells were observed to be fully digested under a microscope (NOPTEC, BDS400). Then, 300 μL of DMEM/F12 medium containing 10% serum (Excell Bio, FND500) was added to terminate the digestion, and 20 μL of resultant was added to a cell counting plate (Countstar, size: 50 pieces/box). The total number of cells was counted with a cell counter (Countstar, I01000). The experimental results are shown in Table 1.
Wherein, “+” indicates that compared with the Basal Medium, the culture medium added with the additive(s) has the effect of promoting the proliferation of two cases of primary ovarian cancer cells isolated from ovarian cancer tissue; “−” indicates that the culture medium added with the additive(s) has the effect of promoting the proliferation of one case of primary ovarian cancer cells isolated from ovarian cancer tissue; “o” indicates that the culture medium added with the additive(s) has no significant effect on the proliferation of at least two cases of primary ovarian cancer cells isolated from ovarian cancer tissue.
According to the above results, factors such as Compound 1, prostaglandin E2, B27, N2, epidermal growth factor, insulin, insulin-transferrin-selenium supplement, amphiregulin, Y-27632, insulin-like growth factor-1, gastrin, cholera toxin were selected for further culture experiments.
The culture mediums for primary ovarian cancer cells containing different combinations of added factors were prepared according to the components in Table 2, to investigate the proliferation-promoting effects of different added factor combinations on primary ovarian cancer cells.
Primary ovarian cancer cells were obtained from ovarian cancer tissue (numbered L46, L55, L56) according to the process of step (2)-3 of Example 1. The obtained cell suspension was divided into 14 equal parts, which were then centrifuged at 1500 rpm for 4 minutes. After centrifugation, the cells were resuspended in 200 μL BM and culture mediums No. 1-13 respectively. The Basal Medium was mixed with matrigel (Corning®, 356231) in a 50:1 ratio on ice. 300 μL of the mixture was plated in a 48-well plate and incubated in a 5% 002 incubator at 37° C. for 25-35 minutes, preferably 30 minutes. The supernatant was discarded. The primary ovarian cancer cells were inoculated into the 48-well plate at a viable cell density of 4×104 cells/well (40,000 cells per well). Each well in the 48-well plate was supplemented to a volume of 1 mL with the corresponding culture mediums, and the resultant was fully mixed. After subjecting to surface sterilization, the plate was placed in a 37° C., 5% CO2 incubator (purchased from Thermo Fisher) for culture.
When the cells grew to more than 85% in the 48-well plate, the culture medium was discarded. The cells were rinsed once with 100 μL of 0.05% trypsin per well (purchased from Gibco). After aspirating the trypsin, 200 μL of 0.05% trypsin was added to each well. The plate was then placed in an incubator at 37° C. and 5% CO2 for reaction of 10 minutes, and the cells were observed to be fully digested under a microscope (CNOPTEC, BDS400). Then, 300 μL of DMEM medium containing 10% fetal bovine serum was added to terminate the digestion, and 20 μL of the resultant was added to a cell counting plate (Countstar, specification: 50 pieces/box). The total number of cells was counted with a cell counter (Countstar, IC1000). The results obtained from primary ovarian cancer cells isolated from L46, L55, and L56 (intraoperative) are shown in
According to the results in
Primary ovarian cancer cells were obtained from tissue samples (numbered A26083, L56, L62) according to the process of step (2)-3 of Example 1. The culture medium with a combination of the effective factors in Example 2 was used for culture. The Basal Medium was mixed with matrigel (Corning®, 356231) in a 50:1 ratio on ice. 4 mL of the mixture was plated in a T12.5 culture flask and incubated in a 5% CO2 incubator at 37° C. for 25-35 minutes, preferably 30 minutes. The supernatant was discarded.
The obtained primary ovarian cancer cells were seeded into a T12.5-well plate (300,000 cells per well) at a viable cell density of 2.4×104 cells/cm2. The plate was then placed in a 37° C., 5% CO2 incubator (purchased from Thermo Fisher) after subjecting to surface sterilization. The cells were cultured and expanded using the culture medium with a combination of effective factors (containing Basal Medium (BM), 20 μM Compound 1, 5 μM prostaglandin E2, 1:100 (v/v) B27, 1:100 (v/v) N2, 10 ng/mL epidermal growth factor, 3 μg/mL insulin, 1:50 (v/v) insulin-transferrin-selenium supplement, 27 ng/mL amphiregulin, 5 μM Y-27632, 50 ng/mL insulin-like growth factor-1, 27 nM gastrin, 0.1 μg/mL cholera toxin) determined in Example 2 until the cells grew to 85% or more, and 500 μL of 0.05% trypsin (purchased from Gibco) was added to rinse the cells for 1 minute. After aspirating the trypsin, 500 μL of 0.05% trypsin was then added to each well, and the plate was placed in a 37° C., 5% CO2 incubator for reaction of 2-10 minutes. The digestion was terminated until the cells were completely digested. After centrifugation at 1500 rpm for 4 minutes, the supernatant was discarded. The cell pellet was resuspended in DMEM/F12, and then 20 μL of the suspension was added to a cell counting plate (manufacturer: Countstar, specification: 50 pieces/box), and the total number of cells was counted with a cell counter (Countstar, IC1000). The obtained cells were used in the following culture experiments.
The following 12 formulations of culture mediums were prepared for experiments.
20 μl of cell resuspension containing 4×104 cells was added to each well, which was then diluted with 1 mL culture medium of the above Formulations 1-12 respectively.
When using the culture medium of Formulation 1, the prepared Compound 1 was added to each well of the 48-well plate seeded with primary cells, with 1 mL per well, and the final concentrations of Compound 1 were 2.5 μM, 5 μM, 10 μM, 20 μM and 40 μM, respectively; and a well of Blank Control (BC) was set using the culture medium of Formulation 1.
When using the culture medium of Formulation 2, the prepared prostaglandin E2 was added to each well of the 48-well plate seeded with primary cells, with 1 mL per well, and the final concentrations of prostaglandin E2 were 2.5 μM, 5 μM, 10 μM, 20 μM and 40 μM, respectively; and a well of Blank Control (BC) was set using the culture medium of Formulation 2.
When using the culture medium of Formulation 3, the prepared B27 was added to each well of the 48-well plate seeded with primary cells, with 1 mL per well, and the final concentrations of B27 were 1:400 (V/V), 1:200 (V/V), 1:100 (V/V), 1:50 (V/V) and 1:25 (V/V), respectively; and a well of Blank Control (BC) was set using the culture medium of Formulation 3.
When using the culture medium of Formulation 4, the prepared N2 was added to each well of the 48-well plate seeded with primary cells, with 1 mL per well, and the final concentrations of N2 were 1:400 (V/V), 1:200 (V/V), 1:100 (V/V), 1:50 (V/V) and 1:25 (V/V), respectively; and a well of Blank Control (BC) was set using the culture medium of Formulation 4.
When using the culture medium of Formulation 5, the prepared epidermal growth factor was added to each well of the 48-well plate seeded with primary cells, with 1 mL per well, and the final concentrations of epidermal growth factor were 2.5 ng/mL, 5 ng/mL, 10 ng/mL, 20 ng/mL and 40 ng/mL, respectively; and a well of Blank Control (BC) was set using the culture medium of Formulation 5.
When using the culture medium of Formulation 6, the prepared insulin was added to each well of the 48-well plate seeded with primary cells, with 1 mL per well, and the final concentrations of insulin were 1 μg/mL, 3 μg/mL, 9 μg/mL, 27 μg/mL and 81 μg/mL, respectively; and a well of Blank Control (BC) was set using the culture medium of Formulation 6.
When using the culture medium of Formulation 7, the prepared insulin-transferrin-selenium supplement was added to each well of the 48-well plate seeded with primary cells, with 1 mL per well, and the final concentrations of insulin-transferrin-selenium supplement were 1:800 (V/V), 1:400 (V/V), 1:200 (V/V), 1:100 (V/V) and 1:50 (V/V), respectively; and a well of Blank Control (BC) was set using the culture medium of Formulation 7.
When using the culture medium of Formulation 8, the prepared amphiregulin was added to each well of the 48-well plate seeded with primary cells, with 1 mL per well, and the final concentrations of amphiregulin were 1 ng/mL, 3 ng/mL, 9 ng/mL, 27 ng/mL and 81 ng/mL, respectively; and a well of Blank Control (BC) was set using the culture medium of Formulation 8.
When using the culture medium of Formulation 9, the prepared Y-27632 was added to each well of the 48-well plate seeded with primary cells, with 1 mL per well, and the final concentrations of Y-27632 were 2.5 μM, 5 μM, 10 μM, 20 μM and 40 μM, respectively; and a well of Blank Control (BC) was set using the culture medium of Formulation 9.
When using the culture medium of Formulation 10, the prepared insulin-like growth factor-1 was added to each well of the 48-well plate seeded with primary cells, with 1 mL per well, and the final concentrations of insulin-like growth factor-1 were 12.5 ng/mL, 25 ng/mL, 50 ng/mL, 100 ng/mL and 200 ng/mL, respectively; and a well of Blank Control (BC) was set using the culture medium of Formulation 10.
When using the culture medium of Formulation 11, the prepared gastrin was added to each well of the 48-well plate seeded with primary cells, with 1 mL per well, and the final concentrations of gastrin were 1 nM, 3 nM, 9 nM, 27 nM and 81 nM, respectively; and a well of Blank Control (BC) was set using the culture medium of Formulation 11.
When using the culture medium of Formulation 12, the prepared cholera toxin was added to each well of the 48-well plate seeded with primary cells, with 1 mL per well, and the final concentrations of cholera toxin were 0.05 μg/mL, 0.1 μg/mL, 0.2 μg/mL, 0.4 μg/mL and 0.8 μg/mL, respectively; and a well of Blank Control (BC) was set using the culture medium of Formulation 12.
When the cells were expanded to about 85% of the 48 wells, the cells were digested and counted, and the proliferation multiple was calculated by referring to the number of cells in the well of Bland Control (BC), and the results were shown in
According to the results in
Using the most preferred concentrations of each added factor in the above culture medium, the culture medium for primary ovarian cancer cells of the invention used in the following examples contains: Basal Medium (BM), 5 μM Compound 1, 2.5 μM prostaglandin E2, 1:100 (V/V) B27, 1:400 (V/V) N2, 10 ng/mL epidermal growth factor, 9 μg/mL insulin, 1:200 (V/V) insulin-transferrin-selenium supplement, 3 ng/mL amphiregulin, 5 μM Y-27632, 50 ng/mL insulin-like growth factor-1, 3 nM gastrin, 0.1 μg/mL cholera toxin (hereinafter referred to as culture medium “OC-2”).
Primary ovarian cancer cells were obtained from 6 cases of intraoperative tissue samples (numbered A26083, L65, L66, L72, L74, L84) according to the process of step (2)-3 of Example 1, and cultured using culture medium OC-2. The Basal Medium was mixed with matrigel (Corning®, 356231) in a 50:1 ratio on ice. 2 mL of the mixture was plated in a 6-well plate and incubated at 37° C. in a 5% CO2 incubator for 25-35 minutes, preferably 30 minutes, and the supernatant was discarded. The obtained primary ovarian cancer cells were seeded into a 6-well plate (250,000 cells per well) at a viable cell density of 2×104 cells/cm2, and 4 mL of culture medium OC-2 was added. After surface sterilization, the plate was placed in a 37° C., 5% CO2 incubator (purchased from Thermo Fisher) for culture.
The cultured primary ovarian cancer cells were observed under the microscope (EVOS M500, Invitrogen).
A cancer tissue of approximately 0.25 cm3 was removed from the intraoperative tissue (sample number L74) of a patient with ovarian cancer and fixed by immersing in 1 mL of 4% paraformaldehyde. The method of Example 4 was used to continue culturing sample L74 to the fourth passage using the culture medium OC-2 for primary ovarian cancer cells of the invention. Primary ovarian cancer cells fixed in 4% paraformaldehyde were embedded in paraffin and cut into 4 μm thick tissue sections using a microtome. Routine immunohistochemical detection was then performed (for specific procedures, see Li et al., Nature Communication, (2018) 9: 2983). The primary antibodies used were ER, PR, P53, NapsinA, Pax-8, WT-1, and Ki-67 (all purchased from CST).
The prior art culture medium (Xuefeng Liu et al., Nat. Protoc., 12(2): 439-451, 2017)), which has the formulation of: DMEM/F12 medium+250 ng/mL amphotericin B (purchased from Selleck)+10 μg/mL gentamicin (purchased from MCE)+0.1 nM cholera toxin+0.125 ng/mL EGF+25 ng/mL hydrocortisone+10 μM Y27632+10% FBS.
Primary ovarian cancer cells were obtained from three cases of ovarian cancer tissue samples (numbered L65, L66, and A22083) according to the process of step (2)-3 of Example 1. The obtained primary ovarian cancer cells were cultured using the prior art culture medium, commercial culture medium and the culture medium OC-2 in Example 3 respectively, and the cells were seeded in a 6-well plate at a viable cell density of 3×104 cells/cm2. After the cells were expanded to 95%, they were digested and counted, and the days in culture until digestion was recorded as a culture cycle. Under this experimental condition, the expanded cells were expanded in different passages. The cells of each passage were digested and counted and the corresponding culture cycle was recorded. PD value was calculated according to the formula, Population Doubling (PD)=3.32*log 10 (total number of cells after digestion/initial number of inoculated cells). For the formula, see Chapman et al., Stem Cell Research & Therapy 2014, 5: 60.
Primary ovarian cancer cells (numbered A22083) were isolated according to the process of step (2)-3 of Example 1, used as a generation, and were cultured with the culture medium OC-2 for primary ovarian cancer cells of the invention until the cells were expanded to 85% and passaged. The cells were passaged and counted according to step (2)-4 in Example 1. The cells were placed in a loading slot (purchased from Corning) at a viable cell density of 1×105 cells/mL. After being thoroughly mixed, they were placed in a 384-well opaque white cell culture plate (purchased from Corning), with a volume of 50 μL per well and a cell number of 5,000 cells/well. The plate was sealed by adding the culture medium for primary ovarian cancer cells of the invention from the edge of the plate, and the sample names, dosing times and testing times of CellTiter-Glo (Promega) were marked on the plate. The surface of the plate was disinfected with 75% alcohol (LIRCON), and the plate was cultured in a 37° C., 5% CO2 incubator, and dosed 24 hours later. Cells of the 1 st, 2nd, 3rd, 4th and 5th passages of culture were respectively obtained for drug screening, and the drug sensitivity of the primary ovarian cancer cells cultured using the culture medium of the invention for continuous passage was tested.
Six drugs (Cytarabine, Doxorubicin, Bortezomib, Panobinostat, Azacitidine, and Homoharringtonine; all purchased from MCE) in 6 concentration gradients were prepared according to the following table, which were added to a 384-well plate (Thermo Fisher) in a volume of 30 μL per well and stored for use.
The plate with prepared drugs was taken out and kept at room temperature. The plate was centrifuged at room temperature for 1 minute at 1,000 rpm in a centrifuge (Beckman), and then taken out from the centrifuge. A high-throughput automated workstation (JANUS, Perkin Elmer) was used for high-throughput dosing. To each well of the 384-well plate with cultured primary ovarian cancer cells was added 0.1 μL of the candidate drugs of corresponding concentrations. After dosing, the surface of 384-well plate was disinfected and moved to a incubator. The cell viability was measured after 72 hours.
CellTiter-Glo luminescent reagent (Promega) was taken out from a 4° C. refrigerator, and 10 mL of the reagent was added into the loading slot. The 384-well plate to be tested was taken out from the incubator, and 10 μL CellTiter-Glo luminescent reagent was added into each well. After being standed for 10 minutes, the test was conducted by using a multifunctional microplate reader (Envision, Perkin Elmer).
According to the formula, cell inhibition rate (%)=100%−chemiluminescence value of the drug-dosed well/chemiluminescence value of the control well*100%, the cell inhibition rate of cells treated with different drugs was calculated, and the half-inhibition rate (IC50) of drugs on cells was calculated by the graphpad prism software. The results are shown in
It can be confirmed from
The invention provides a primary cell culture medium and a culture method for culturing primary ovarian cancer cells in vitro, and the cultured cells can be used for efficacy evaluation and screening of drugs. Therefore, 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 |
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202111201798.2 | Oct 2021 | CN | national |
Filing Document | Filing Date | Country | Kind |
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PCT/CN2021/131967 | 11/22/2021 | WO |