CN115975934A - Culture medium, culture method and application of ovarian cancer organoid - Google Patents
Culture medium, culture method and application of ovarian cancer organoid Download PDFInfo
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Abstract
The invention provides a culture medium and a culture method for ovarian cancer organoids. The medium comprises an MST1/2 kinase inhibitor; b27 additives; an N2 additive; insulin-transferrin-selenium supplement; insulin; an epidermal cytokine; fetal bovine serum; basic fibroblast growth factor; fibroblast growth factor 7; rho protease inhibitors. Compared with the existing culture medium and culture mode, the in vitro culture using the culture medium has higher amplification efficiency; the culture medium is used for culturing the ovarian cancer organoid, can maintain the morphological structure and pathological characteristics of primary tissues, improve the success rate and survival rate of culturing the ovarian cancer organoid, and provide a research basis for the personalized treatment of patients.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a culture medium for culturing ovarian cancer organoids, a method for culturing ovarian cancer organoids by using the culture medium, and application of the culture medium in curative effect evaluation and screening of medicines.
Background
Ovarian cancer refers to a malignant tumor disease occurring in the ovary, is one of the common malignant tumors of female reproductive organs, and has the incidence rate second to cervical cancer and uterine corpus cancer. Ovarian cancer is the most common of epithelial cancers, and is followed by malignant germ cell tumors, wherein the death rate of ovarian epithelial cancers accounts for the first part of various gynecological tumors, and can cause serious threat to the life of women. Ovarian cancer is asymptomatic in early stage, digestive tract symptoms such as lower abdominal discomfort, abdominal distension, appetite reduction and the like can appear in late stage, the main treatment modes comprise surgical resection, drug treatment and radiation treatment, and the overall prognosis is poor.
Two-dimensional cell culture is mostly adopted in the traditional clinical drug sensitivity detection. However, the two-dimensional cultured cells only simulate the tissue physiological conditions to a limited extent, lack the real tissue structure in vivo, easily cause low differentiation level and loss of cell physiological functions, and further cause the obtained experimental results to be difficult to predict the clinical practical results. Organoids, belonging to three-dimensional (3D) cell cultures, are mainly derived from human embryonic stem cells, induced pluripotent stem cells and adult stem cells with differentiation capacity. 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 miniature 3D tumor cell models grown in the laboratory from primary tumors taken from patients. 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, R-spondin-1, noggin and some expensive protein factors are mostly adopted in the ovarian cancer organoid culture method, so that the organoid culture cost is high; and the technical operation is complex and the technical difficulty is high, so that the large-scale commercial application of the technology is limited. Therefore, there is a need to develop a low-cost, simple organoid culture method and medium with high success rate.
Disclosure of Invention
In order to solve the technical problems, the invention provides a culture medium and a culture method for rapidly amplifying ovarian cancer organoids in vitro.
One aspect of the invention provides a culture medium for an ovarian cancer organoid, said culture medium comprising an inhibitor of MST1/2 kinase; b27 additives; an N2 additive; insulin-transferrin-selenium supplement; insulin; an epidermal cytokine; fetal bovine serum; basic fibroblast growth factor; fibroblast growth factor 7; and a Rho protease inhibitor selected from at least one of Y27632, fasudil, and H-1152. Wherein the MST1/2 kinase inhibitor comprises a compound of formula (I) or a pharmaceutically acceptable salt, or solvate thereof,
wherein, the first and the second end of the pipe are connected with each other,
R 1 selected from C1-C6 alkyl, C3-C6 cycloalkyl, C4-C8 cycloalkylalkyl, C2-C6 spirocycloalkyl, and optionally substituted with 1-2 independent R 6 Substituted aryl (e.g., phenyl, naphthyl, and the like), arylC 1-C6 alkyl (e.g., benzyl, and the like), and heteroaryl (e.g., thienyl, and the like);
R 2 and R 3 Each independently selected from C1-C6 alkyl, preferably C1-C3 alkyl, more preferably methyl;
R 4 and R 5 Each independently selected from hydrogen, C1-C6 alkyl, C3-C6 cycloalkyl, C4-C8 cycloalkylalkyl, C1-C6 alkylhydroxy, C1-C6 haloalkyl, C1-C6 alkylaminoC 1-C6 alkyl, C1-C6 alkoxyC 1-C6 alkyl, and C3-C6 heterocycloC 1-C6 alkyl (said heterocyclyl is selected from, for example, piperidinyl, tetrahydropyranyl, and the like);
R 6 selected from the group consisting of halogen (preferably fluorine and chlorine, more preferably fluorine), C1-C6 alkyl (preferably methyl), C1-C6 alkoxy (preferably methoxy), and C1-C6 haloalkyl (preferably trifluoromethyl).
In a preferred embodiment, the MST1/2 kinase inhibitor comprises a compound of formula (Ia) or a pharmaceutically acceptable salt, or solvate thereof,
wherein the content of the first and second substances,
R 1 selected from C1-C6 alkyl, optionally substituted with 1-2 independent R 6 Substituted phenyl, optionally substituted with 1-2 independent R 6 Substituted thienyl, and optionally substituted with 1-2 independent R 6 Substituted benzyl, R 1 More preferably optionally substituted with 1-2 independent R 6 Substituted phenyl;
R 5 selected from hydrogen, C1-C6 alkyl, and C3-C6 cycloalkyl, R 5 More preferably hydrogen;
R 6 each independently selected from halogen, C1-C6 alkyl, and C1-C6 haloalkyl, R 6 More preferably fluorine, methyl or trifluoromethyl.
Preferably, the MST1/2 inhibitor is at least one selected from the following compounds or a pharmaceutically acceptable salt, or solvate thereof.
Most preferably, the MST1/2 kinase inhibitor of the invention is compound 1.
Preferably, the Rho protein kinase inhibitor of the invention is Y27632.
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 range of the MST1/2 kinase inhibitor is usually 2.5-40 μ M, and more preferably 5-20 μ M;
(2) The volume ratio of the B27 additive to the culture medium is typically in the range of 1: 25-1: 400, more preferably 1:50 to 1:100, respectively;
(3) The volume ratio of the N2 additive to the culture medium is typically in the range of 1:100 to 1:800, more preferably 1: 200-1: 800;
(4) The concentration range of the epidermal cytokine is usually 2.5-10 ng/mL;
(5) The concentration range of the insulin is usually 0.25 to 4 mug/mL, and more preferably 0.5 to 1 mug/mL;
(6) The volume ratio of the insulin-transferrin-selenium supplement to the culture medium is generally in the range of 1;
(7) The concentration range of the Rho protein kinase inhibitor is usually 2.5 to 40. Mu.M, more preferably 5 to 10. Mu.M;
(8) The volume ratio of the fetal calf serum to the culture medium is usually in the range of 1.25% (v/v) to 20% (v/v), more preferably 5% (v/v) to 20% (v/v);
(9) The concentration range of the basic fibroblast growth factor is usually 2.5-20 ng/mL, and more preferably 2.5-10 ng/mL;
(10) The concentration of the fibroblast growth factor 7 is usually in the range of 2.5 to 20ng/mL, more preferably 5 to 20ng/mL.
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 streptomycin concentration ranges from 25 to 400. Mu.g/mL when the antibiotic is selected from streptomycin/penicillin, the penicillin concentration 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.
According to a second aspect, the invention also provides a method of culturing ovarian cancer organoids in vitro. In the method for culturing ovarian cancer organoids in vitro, primary ovarian cancer cells are cultured in vitro by using the ovarian cancer organoid culture medium of the present invention.
The ovarian cancer organoid culture method comprises the following steps:
1. separating a sample from ovarian cancer solid tumor tissue to obtain ovarian cancer primary cells. The process includes the following steps.
(1) Separating the ovarian cancer tissue sample, adding a basic culture medium and tissue digestive juice (the adding amount of the tissue digestive juice is 5-10mL for 1g of tumor tissue) according to the volume ratio of 1:3, placing the mixture in a constant temperature shaking table for digestion, wherein the digestion temperature range is 4-37 ℃, and the digestion rotation speed range is 200-350 rpm;
(2) The digestion can be stopped until no obvious tissue block is found, and the digestion time is 3 to 6 hours;
(3) And removing supernatant after centrifugation, wherein the centrifugation rotating speed range is 1200-1600 rpm, the centrifugation time is 2-6 minutes, and a basic culture medium is added for re-suspension for later use.
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. Culturing Using the ovarian cancer organoid Medium of the present invention
Resuspending and counting the ovarian cancer primary cells obtained in the step 1 by using a basal culture medium, and uniformly mixing the ovarian cancer primary cells and the matrigel ice according to the volume ratio of 1:1, wherein the final cell density is 1-8 multiplied by 10 5 Taking matrigel and cell suspension to form coagulated liquid drops in a culture plate, standing the culture plate for 10-60 minutes at 4-37 ℃ until the matrigel is completely coagulated, adding the ovarian cancer organoid culture medium, and culturing in an incubator.
The present invention also provides a method for evaluating or screening a drug for treating ovarian cancer disease, comprising the steps of:
(1) Culturing the ovarian cancer organoid by using the culture method of the ovarian cancer 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 technical scheme of the invention can achieve the following technical effects:
(1) The success rate of culturing the ovarian cancer organoid is improved and reaches over 90 percent;
(2) Ensuring that the ovarian cancer organoid subjected to in vitro primary culture can keep the pathological characteristics of a patient;
(3) The amplification efficiency is high, the ovarian cancer organoids can be quickly cultured, and the amplified ovarian cancer organoids can be continuously passed;
(4) The culture cost is controllable, and expensive factors such as Wnt agonist, R-spondin family protein, noggin protein, BMP inhibitor, fibroblast growth factor 10 (FGF 10) and the like do not need to be added into the culture medium;
(5) The ovarian cancer organoids obtained by the culture of the technology have large quantity, and are suitable for screening candidate compounds at high flux and providing high-flux drug in-vitro sensitivity function tests for patients.
Drawings
FIG. 1 is a graph showing the effect of different combinations of factors added to ovarian cancer organoid culture media on ovarian cancer organoid growth.
FIGS. 2A-2J are graphs showing the effect of different concentrations of an ovarian cancer organoid medium with added factor on ovarian cancer organoid growth.
FIGS. 3A-3F are photographs of a microscope showing observation of ovarian cancer organoids cultured using the ovarian cancer organoid medium of the present invention.
FIGS. 4A-4G show immunohistochemical results for primary ovarian cancer tissue cells.
FIGS. 5A-5G are immunohistochemical results of culturing primary ovarian cancer tissue cells to third generation ovarian cancer organoids using ovarian cancer organoid culture medium of the present invention.
FIG. 6 is a photomicrograph of an ovarian cancer organoid obtained by culturing ovarian cancer primary cells using the ovarian cancer organoid medium of the present invention and a literature culture medium, respectively.
FIGS. 7A and 7B show the results of drug susceptibility testing of ovarian cancer organoids cultured using the ovarian cancer organoid medium of the present invention, wherein FIG. 7A shows photographs of organoid growth without drug treatment and 3 days after drug treatment; FIG. 7B shows a graph of drug sensitivity to ovarian cancer organoid growth for various concentrations of test drug.
Detailed Description
For a better understanding of the present invention, the present invention will be further described with reference to the following examples and the accompanying drawings, which are illustrative of the present invention and are not to be construed as limiting the present invention.
[ 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): 2-amino-2- (2,6-difluorophenyl) acetic acid (2.0 g) was added to a round bottom flask followed by methanol (30 ml) and then thionyl chloride (1.2 ml) was added dropwise under ice-bath. The reaction system was allowed to react overnight at 85 ℃. After the reaction was completed, the solvent was evaporated to dryness under reduced pressure to obtain a white solid, which was used in the next step.
Methyl 2- ((2-chloro-5-nitropyrimidin-4-yl) amino) -2- (2,6-difluorophenyl) acetate (A3): to a round bottom flask was added methyl 2-amino-2- (2,6-difluorophenyl) acetate (2 g), followed by acetone (30 ml) and potassium carbonate (2.2 g), then the system was cooled to-10 ℃ with an ice salt bath, followed by the slow addition of 2,4-dichloro-5-nitropyrimidine (3.1 g) in acetone. The reaction was stirred at room temperature overnight. After the reaction, the reaction mixture was filtered, the solvent was removed from the filtrate under reduced pressure, and the residue was purified by pressure silica gel column chromatography to obtain Compound A3.LC/MS: m + H359.0.
2-chloro-7- (2,6-difluorophenyl) -7,8-dihydropteridin-6 (5H) -one (A4): to a round bottom flask was added methyl 2- ((2-chloro-5-nitropyrimidin-4-yl) amino) -2- (2,6-difluorophenyl) acetate (2.5 g) followed by acetic acid (50 ml) and iron powder (3.9 g). The reaction was stirred at 60 ℃ for two hours. After the reaction was completed, the solvent was evaporated to dryness under reduced pressure, and the resultant 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 in analogy to compound 1 and their structural and mass spectral data are shown in the table below.
Example 1 Effect of factors added to ovarian cancer organoid Medium on growth of ovarian cancer organoids
(1) Preparation of ovarian cancer organoid culture medium
First, a basal medium containing an initial medium is prepared. The starting medium may be selected from DMEM/F12, DMEM, F12 or RPMI-1640, commonly used in the art. In this example, the basic medium formulation is: DMEM/F12 medium (from Corning) + 100. Mu.g/mL Primocin (from InvivoGen, 0.2% (v/v), commercial product concentration 50 mg/mL). Different additives (see table 1) are added into the basic culture medium respectively to prepare the ovarian cancer organoid culture medium containing different additive components.
(2) Isolation and processing of ovarian cancer primary cells
1 sample selection
Ovarian cancer solid tumor tissue samples (intraoperative/endoscopic) were obtained from patients by medical professionals at a medical professional institution, all signed with informed consent. Intraoperative sample 0.25cm 3 Endoscope sample 0.025cm 3 (ii) a And is stored and transported by using a commercial tissue preservation solution (manufacturer: miltenyi Biotec).
2 Material preparation
And (3) sterilizing the surfaces of a 15mL sterile centrifuge tube, a pipette, a 10mL pipette, a sterile gun head and the like, and then placing the sterilized surfaces into a super clean bench for ultraviolet irradiation for 30 minutes. Basal medium was removed from the 4 ℃ freezer 30 minutes earlier and tissue digestate was removed from the-20 ℃ freezer 30 minutes earlier (recipe below).
Tissue digestive juice: 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), BSA (10 mg/mL).
Collagenase ii, collagenase iv, dnase, and hyaluronidase mentioned above were all purchased from Sigma company; calcium chloride was purchased from bio-engineering (shanghai) gmbh; BSA was purchased from Biofrox.
3. Isolation of Primary ovarian cancer cells
3.1 taking tissue sample in a culture dish in a super clean bench, removing the tissue with blood, washing with a basic culture medium for 2 times, transferring the tissue to another culture dish, mechanically separating with a sterile scalpel, and dividing the tissue block into 1 × 1 × 1mm 3 Size;
3.2 sucking the cut intraoperative or endoscopic tissues into a 15mL centrifuge tube, adding 5mL of basal medium, uniformly mixing, and centrifuging at 1500rpm for 4 minutes;
3.3 discarding the supernatant, adding a basal medium and a tissue digestive juice according to a proportion of 1:3 (note: the adding amount of the tissue digestive juice is about 10mL for 1g of tumor tissue), marking the name and the number of the sample, sealing the sample by a sealing film, digesting in a shaker (ZQLY-180N) at 300rpm at 37 ℃, observing whether the digestion is completed every 30 minutes, and judging that no particles visible to naked eyes exist, wherein the digestion time is about 4 hours;
3.4 after the digestion is finished, filtering out undigested tissue agglomerates by a 100-micron filter screen, flushing the tissue agglomerates on the filter screen into a centrifuge tube by using a basic culture medium to reduce cell loss, and centrifuging at 1500rpm for 4 minutes at 25 ℃;
3.5 abandoning the supernatant, observing whether blood cells exist, if the blood cells exist, adding 8mL of blood cell lysate (purchased from Sigma company), mixing uniformly, cracking at 4 ℃ for 20 minutes, reversing and mixing uniformly for once, and centrifuging at 25 ℃ at 1500rpm for 4 minutes;
3.6 discard the supernatant, add 2mL of basal medium to resuspend the cells for further use.
4 cell counting and processing
4.1 Observation under a lens: a small amount of the resuspended cells are removed and spread in a culture dish, and the density and the 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 (Biotechnology (Shanghai) Co., ltd.) were mixed well, 20. Mu.L of the mixture was added to a cell counting plate (Countstar, specification: 50 plates/box), and the percentage of viable large cells (cell size >10 μm) = viable cell count/total cell count x 100% was calculated under a cell counter (Countstar, IC 1000).
(3) Culture of ovarian cancer organoids
Resuspending and counting ovarian cancer primary cells isolated from 2 ovarian cancer tissues (numbers L55 and L56) according to the above step (2) in a basal medium, and mixing the cells with matrigel (according to the volume ratio of 1:1: (1))
356231 ) mixed well on ice with a final cell density of 5X 10 5 And (4) taking 7 mu L of matrigel and cell suspension to form a solidified drop in the center of each well of a 96-well culture plate, and standing the culture plate at 37 ℃ for 30 minutes until the matrigel is completely solidified. The different components of the medium in Table 1 were added to 96-well plates in 100. Mu.L/well volumes. As an experimental control, a basal medium to which no additive component was added was used. After 7 to 10 days of culture, 50. Mu.L of CellTiter-Glo (available from Promega) luminescent reagent was added to each well, and after standing for 10 minutes, the mixture was mixed and detected by a multifunctional microplate reader (Envision, perkinelmer). The relative cell viability (%) = test well chemiluminescence value/control well chemiluminescence value x 100% was calculated according to the formula, and the effect of different additives on the growth of ovarian cancer organoids was presumed. The experimental results are shown in table 1.
TABLE 1 additive ingredients in culture media and cell proliferation promoting effects
Wherein "+" indicates that the medium added with the additive has the function of promoting the proliferation of at least two cases of ovarian cancer primary cells separated from ovarian cancer tissues compared with a basic medium; "-" indicates that the medium to which the additive was added exhibited an effect of promoting proliferation of one example of ovarian cancer primary cells isolated from ovarian cancer tissue; ". Smallcircle" indicates that the medium to which the additive was added had no significant effect on the proliferation of primary ovarian cancer cells isolated from ovarian cancer tissue.
Based on the above results, compounds 1, B27, N2, epidermal cytokine, insulin-transferrin-selenium supplement, Y-27632, fetal bovine serum, basic fibroblast growth factor, forsklin, fibroblast growth factor 7, cholera toxin and other additives were selected and further subjected to culture experiments.
Example 2 Effect of combinations of different additional factors in ovarian cancer organoid Medium on ovarian cancer organoid growth
Ovarian cancer organoid culture media with different additive factor combinations are prepared according to the components in the table 2, and the growth promoting effect of the different additive factor combinations on ovarian cancer organoids is examined.
TABLE 2 preparation of the media of different compositions (final concentration)
Obtaining ovarian cancer primary cells from ovarian cancer tissues (numbered L53, L57, L58) according to the method of step 3 of step (2) of example 1, resuspending and counting in basal medium, and comparing with matrigel according to the volume ratio of 1:1: (
356231 ) mixed well on ice with a final cell density of 5X 10 5 And (4) taking 20 mu L of matrigel and the cell suspension to form a coagulated droplet at the center of each well of a 48-well culture plate, and standing the culture plate at 37 ℃ for 30 minutes until the matrigel is completely coagulated. Finally adding BM culture medium and 1mL to 48-well plates of culture medium corresponding to No. 1-No. 13 respectively. After culturing for 7-10 days, after organoids are obviously formed, 20 μ L of CellTiter-Glo (purchased from Promega corporation) luminescent reagent is added into each hole, and the mixture is uniformly mixed after standing for 10 minutes and is detected by using a multifunctional microplate reader (Envision of Perkinelmer). And (4) calculating the relative activity rate (%) = experimental hole chemiluminescence value/control hole chemiluminescence value multiplied by 100% according to a formula cell, and obtaining the ovarian cancer organoid growth promoting effect of different component culture media. The experimental results are shown in fig. 1.
From the results shown in FIG. 1, it is clear that the proliferation of ovarian cancer primary cells can be promoted to a different extent in any of the above-mentioned culture media Nos. 1 to 13, compared with the basal medium. When the addition factors Forsklin (No. 11) and cholera toxin (No. 13) are omitted, the proliferation promoting effect of the culture medium formula is more obvious. Thus, in the examples that follow, further studies were conducted using factors including compound 1, B27, N2, epidermal cytokine, insulin-transferrin-selenium supplement, Y-27632, fetal bovine serum, basic fibroblast growth factor, fibroblast growth factor 7, etc., as a medium formulation for culturing ovarian cancer organoids.
EXAMPLE 3 proliferation of ovarian cancer organoids by different concentrations of factors added to ovarian cancer organoid culture Medium
Ovarian cancer primary cells were obtained from ovarian cancer tissue (accession number L74) as in step (2) 3 of example 1, resuspended and counted in basal medium, at a volume ratio of 1:1 to matrigel (K: (K) (K))
356231 ) mixed well on ice with a final cell density of 5X 10 5 Taking 7 mu L of matrigel and cell suspension to form a coagulated liquid drop at the center of each well of a 96-well culture plateThe plate was allowed to stand at 37 ℃ for 30 minutes until the matrigel was completely solidified, and was used for the following culture experiment.
Next, an experiment was performed by preparing an ovarian cancer organoid medium containing basal medium BM, 20. Mu.M Compound 1, 1 (v/v) B27, 1 (v/v) N2, 10ng/mL epidermal cytokine, 1. Mu.g/mL insulin, 1 (v/v) insulin-transferrin-selenium supplement, 10. Mu. M Y-27632, 10% (v/v) fetal bovine serum, 10ng/mL basic fibroblast growth factor, 5ng/mL fibroblast growth factor 7, and the following 10 formulation media.
Formula 1: the ovarian cancer organoid culture medium does not contain the compound 1;
and (2) a formula: the ovarian cancer organoid culture medium does not contain B27;
and (3) formula: the components of the ovarian cancer organoid culture medium do not contain N2;
and (4) formula 4: the ovarian cancer organoid culture medium does not contain epidermal cytokines;
and (5) formula: the ovarian cancer organoid culture medium component is free of insulin;
and (6) formula: the ovarian cancer organoid culture medium composition does not contain insulin-transferrin-selenium supplement;
and (3) formula 7: the components of the ovarian cancer organoid culture medium do not contain Y-27632;
and (4) formula 8: the components of the ovarian cancer organoid culture medium do not contain fetal calf serum;
formula 9: the components of the ovarian cancer organoid culture medium do not contain alkaline fibroblast growth factors;
formula 10: the components of the ovarian cancer organoid culture medium do not contain fibroblast growth factor 7;
when the culture medium of formula 1 was used, 100. Mu.L of the prepared compound 1 was added to each well of a 96-well plate inoculated with primary cells, and the final concentrations of the compound 1 were 2.5. Mu.M, 5. Mu.M, 10. Mu.M, 20. Mu.M, and 40. Mu.M, respectively; and control wells (BC) were set using medium of formula 1.
When the medium of formulation 2 was used, 100 μ L of B27 prepared per well was added to a 96-well plate inoculated with primary cells, and the volume ratios of B27 to the medium were 1; and control wells (BC) were set using medium of formula 2.
When the medium of formulation 3 was used, 100 μ L of prepared N2 was added to each well of a 96-well plate inoculated with primary cells, and the volume ratios of N2 to the medium were 1; and control wells (BC) were set using medium of formula 3.
When the culture medium of formula 4 is used, 100 μ L of the prepared epidermal cytokine is added to a 96-well plate inoculated with primary cells, and the final concentrations of the epidermal cytokine are respectively 2.5ng/mL, 5ng/mL, 10ng/mL, 20ng/mL and 40ng/mL; and control wells (BC) were set using medium of formula 4.
When the culture medium of formula 5 is used, 100. Mu.L of prepared insulin is added to a 96-well plate inoculated with primary cells, and the final concentrations of the insulin are respectively 0.25. Mu.g/mL, 0.5. Mu.g/mL, 1. Mu.g/mL, 2. Mu.g/mL and 4. Mu.g/mL; and control wells (BC) were set using medium of formula 5.
In the case of the culture medium of formulation 6, 100 μ L of the formulated insulin-transferrin-selenium supplement per well was added to the 96-well plate inoculated with the primary cells, respectively, in a volume ratio of 1; and control wells (BC) were set using medium of formula 6.
When the culture medium of formula 7 is used, 100. Mu.L of prepared Y-27632 is added to a 96-well plate inoculated with primary cells, and the final concentrations of Y-27632 are 2.5. Mu.M, 5. Mu.M, 10. Mu.M, 20. Mu.M and 40. Mu.M respectively; and control wells (BC) were set using medium of formulation 7.
When the medium of formulation 8 was used, 100. Mu.L of prepared fetal bovine serum was added to each well of a 96-well plate in which primary cells were inoculated, and the volume ratios of fetal bovine serum to the medium were 1.25% (v/v), 2.5% (v/v), 5% (v/v), 10% (v/v), and 20% (v/v), respectively; and control wells (BC) were set using medium of formula 8.
When the culture medium of formula 9 is used, 100 μ L of prepared basic fibroblast growth factor is added to a 96-well plate inoculated with primary cells, and the final concentrations of the basic fibroblast growth factor are respectively 2.5ng/mL, 5ng/mL, 10ng/mL, 20ng/mL and 40ng/mL; and control wells (BC) were set using medium of formula 9.
When the culture medium of the formula 10 is used, 100 mu L of prepared fibroblast growth factor 7 is respectively added into a 96-well plate inoculated with primary cells, and the final concentrations of the fibroblast growth factor 7 are respectively 2.5ng/mL, 5ng/mL, 10ng/mL, 20ng/mL and 40ng/mL; and control wells (BC) were set using medium of formula 10.
After organoids were cultured for 7-10 days, relative proliferation fold was calculated with reference to the number of cells in control wells (BC), respectively, and the results are shown in fig. 2A to 2J, respectively. In FIGS. 2A to 2J, the relative proliferation factor is the ratio of the proliferation factor of the ovarian cancer organoid after 7 to 10 days of culture using each culture medium to the proliferation factor of the ovarian cancer organoid after 7 to 10 days of culture using the corresponding control well. The ratio is more than 1, which indicates that the proliferation promoting effect of the prepared culture medium containing factors or small molecular compounds with different concentrations is better than that of a control 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 FIGS. 2A to 2J, the concentration of Compound 1 is preferably in the range of 2.5 to 40. Mu.M, more preferably 5 to 20. Mu.M; the volume ratio of the B27 additive to the culture medium is preferably in the range of 1; the volume ratio of the N2 additive to the culture medium is preferably 1; the preferred concentration range of the epidermal cell factor is 2.5-10 ng/mL; the content of insulin is preferably 0.25 to 4. Mu.g/ml, more preferably 0.25 to 1. Mu.g/ml; the volume ratio of the insulin-transferrin-selenium supplement to the culture medium is preferably in the range of 1; the content of Y-27632 is preferably 2.5-40 μ M, more preferably 5-10 μ M; the volume content of the fetal bovine serum is preferably 1.25 to 20% (v/v), more preferably 5 to 20% (v/v); the content of the basic fibroblast growth factor is preferably 2.5-20 ng/ml, and more preferably 2.5-10 ng/ml; the content of fibroblast growth factor 7 is preferably 2.5-20 ng/ml, more preferably 5-20 ng/ml.
The ovarian cancer organoid culture medium of the present invention, used in the following examples, contains: basal medium BM, 10. Mu.M Compound 1, 1.
Example 4 ovarian cancer organoid culture
Ovarian cancer primary cells (L65, L66, L70, L74, L82, L84) were obtained as described in example 1 (2), resuspended in basal medium and counted in a volume ratio of 1:1 to matrigel (M) ((L65), L66, L70, L74, L82, L84)
356231 ) mixed well on ice with a final cell density of 5X 10 5 and/mL, 50. Mu.L of matrigel was taken together with the cell suspension to form a coagulated droplet at the center of each well of the 24-well plate, the plate was left to stand at 37 ℃ for 30 minutes until the matrigel was completely coagulated, and 1mL of the ovarian cancer organoid medium OC-3 of the present invention at room temperature was gently added to each well along the well side wall using a pipette. Sterilizing the surface of 24-well culture plate, placing at 37 deg.C, 5% 2 Incubators (purchased from Saimeri fly) for cultivation. After culturing for 4 to 10 days, the cultured ovarian cancer organoids were observed with a microscope (Invitrogen, EVOS M500), and FIGS. 3A to 3F are photographs of the ovarian cancer organoids taken under a 10-fold objective lens. The ovarian cancer organoids are regularly spherical under the microscope, and the surface is smooth.
EXAMPLE 5 organotypic identification of cultured ovarian cancer
About 0.25cm was taken from the intraoperative tissue (sample No. L74) of one ovarian cancer patient 3 The size of the cancer tissue was fixed by immersion in 1mL of 4% paraformaldehyde. Sample L74 was maintained using the ovarian cancer organoid medium of the present invention using the method of example 3Culturing until 3 rd generation. The 4% paraformaldehyde fixed ovarian cancer organoids were paraffin embedded and sliced into 4 μm thick tissue sections with a microtome. Conventional immunohistochemical detection was then performed (see, for specific steps, li et al, nature Communication, (2018) 9. The primary antibodies used were ER, PR, P53, napsinA, pax-8, WT-1, ki-67 (all available from CST).
FIGS. 4A-4G and 5A-5G are a comparison of immunohistochemical results for primitive tissues and ovarian cancer organoids obtained by culturing the primitive tissue cells using the ovarian cancer organoid culture medium OC-3 of the present invention, respectively. Wherein, fig. 4A and 5A are respectively a picture of a labeled ER antibody of an ovarian cancer tissue and an ovarian cancer organoid after culture, fig. 4B and 5B are respectively a picture of a labeled PR antibody of an ovarian cancer tissue and an ovarian cancer organoid after culture, fig. 4C and 5C are respectively a picture of a labeled P53 antibody of an ovarian cancer tissue and an ovarian cancer organoid after culture, fig. 4D and 5D are respectively a picture of a labeled napsin a antibody of an ovarian cancer tissue and an ovarian cancer organoid after culture, fig. 4E and 5E are respectively a picture of a labeled Pax-8 antibody of an ovarian cancer tissue and an ovarian cancer organoid after culture, fig. 4F and 5F are respectively a picture of a labeled WT-1 antibody of an ovarian cancer tissue and an ovarian cancer organoid after culture, and fig. 4G and 5G are respectively a picture of a labeled Ki-67 antibody of an ovarian cancer tissue and an ovarian cancer organoid after culture. From this, it was confirmed that, when the ovarian cancer organoids cultured by the technique of the present invention were cultured up to the 3 rd generation, the expression of the ovarian cancer-associated biomarker on the ovarian cancer organoids substantially matched the expression of the biomarker in the original tissue sections from which the ovarian cancer organoids originated. This demonstrates that ovarian cancer organoids cultured using the present technology retain the original pathological properties of ovarian cancer patient cancer tissue.
Example 5 comparison of culture Effect with literature Medium
(1) Preparation of culture Medium
Literature culture medium (odd Kopper et al, nature media, 2019, 838-849): DMEM/F12+25% (v/v) Wnt3A (from Invitrogen) +1% penicillin/streptomycin + 1.
(2) Acquisition of ovarian cancer primary cells and culture of ovarian cancer organoids
Ovarian cancer primary cells were obtained from intraoperative tissue samples (number L68) according to the method of step (2) 3 of example 1, resuspended and counted in basal medium, and mixed with matrigel (v) at a volume ratio of 1:1
356231 ) mixed well on ice with a final cell density of 5X 10 5 and/mL, 50. Mu.L of matrigel and cell suspension were taken to form a coagulated droplet at the center of each well of a 24-well plate, the plate was left to stand at 37 ℃ for 30 minutes until the matrigel was completely coagulated, and 1mL of the ovarian cancer organoid medium OC-3 of the present invention and the literature medium at room temperature was gently added to each well along the side wall of the well using a pipette. Sterilizing the surface of 24-well culture plate, placing at 37 deg.C, 5% 2 Incubators (purchased from Saimeri fly) for culture. After 7 days of culture, the cultured ovarian cancer primary cells were observed using a microscope (EVOS M500, invitrogen), and FIG. 6 shows the ovarian cancer organoids recorded under a 10-fold objective lens.
From the results of FIG. 6, it is clear that when ovarian cancer organoids are cultured in the ovarian cancer organoid culture medium of the present invention in vitro, the number of organoids is large and organoid structure is intact, compared to the literature culture medium, and the effect is significantly superior to the literature culture medium.
EXAMPLE 6 ovarian cancer organoids expanded using the culture Medium of the present invention for drug screening
(1) Ovarian cancer organoid culture
Ovarian cancer primary cells were isolated from an ovarian cancer intraoperative sample (L74) by the method of (2) in example 1, and organoid culture was performed using OC-3 medium, and drug screening was performed when the ovarian cancer organoid diameter exceeded 50 μm.
(2) Screening drug formulations
2 drugs (carboplatin, gemcitabine; both from MCE) were formulated in 5 concentration gradients according to the following table and stored until use.
TABLE 3 drug action concentration settings
(3) Dosing
Taking out the prepared medicine, placing at room temperature, and preparing the medicine with OC-3 culture medium for use. The organoids obtained by culturing according to step (1) were removed from the incubator, the culture medium in the culture well was removed, and 400. Mu.L of the culture medium containing the drug was gradually introduced into the culture well along the walls of the well. After the dosing is finished, the surface of the 48-hole plate is disinfected and then is transferred to an incubator to be cultured continuously, and the organoid activity is measured after 3 days.
(4) Organoid viability assay
CellTiter-Glo luminescence reagent (purchased from Promega) was taken out from a refrigerator at 4 ℃, 48-well plates to be detected were taken out from an incubator, 50. Mu.L of CellTiter-Glo luminescence reagent was added to each well, and the mixture was left to stand for 30 minutes, mixed and detected using a multifunctional microplate reader (Envision, perkin Elmer).
(5) Data processing
The relative activity rates of the different drugs were calculated according to the formula relative activity rate (%) = (chemiluminescence numerical drug treatment group in culture well on the third day/chemiluminescence numerical drug treatment group in culture well on the zeroth day) × 100%, and the results are shown in fig. 7A and 7B. Fig. 7A is a photograph of organoid growth without drug treatment and after 3 days of drug treatment taken by a 4-fold objective microscope (EVOS M500, invitrogen) and fig. 7B is a graph of drug sensitivity to ovarian cancer organoid growth for various concentrations of test drug.
From FIGS. 7A and 7B, it was confirmed that the ovarian cancer organoids cultured using the organoid medium OC-3 of the present invention were screened for drugs, and from the results, the effectiveness of the drugs in clinical use in patients with ovarian cancer could be judged.
Industrial applicability
The invention provides a culture medium and a culture method for culturing ovarian cancer organoids, which can be used for evaluating and screening the curative effect of medicaments 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 (9)
1. A culture medium for an ovarian cancer organoid, comprising an MST1/2 kinase inhibitor; b27 additives; an N2 additive; insulin-transferrin-selenium supplement; insulin; an epidermal cytokine; fetal bovine serum; basic fibroblast growth factor; fibroblast growth factor 7; and at least one Rho protease inhibitor selected from the group consisting of Y27632, fasudil, and H-1152,
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,
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:
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:
(1) The concentration of the MST1/2 kinase inhibitor is 2.5-40 mu M;
(2) The volume ratio of the B27 additive to the culture medium is 1: 25-1: 400;
(3) The volume ratio of the N2 additive to the culture medium is 1:100 to 1:800;
(4) The concentration of the epidermal cell factor is 2.5-10 ng/mL;
(5) The concentration of the insulin is 0.25-4 mug/mL;
(6) The volume ratio of the insulin-transferrin-selenium supplement to the culture medium is 1;
(7) The concentration of the Rho protein kinase inhibitor is 2.5-40 mu M;
(8) The volume ratio of the fetal calf serum to the culture medium is 1.25% (v/v) to 20% (v/v);
(9) The concentration of the basic fibroblast growth factor is 2.5-20 ng/mL;
(10) The concentration of the fibroblast growth factor 7 is 2.5-20 ng/mL.
7. The culture medium according to any one of claims 1 to 5, further comprising:
a starting medium selected from DMEM/F12, DMEM, F12 or RPMI-1640; and an antibiotic selected from one or more of streptomycin/penicillin, amphotericin B, and Primocin.
8. A method for culturing an ovarian cancer organoid, comprising the steps of:
(1) Preparing a culture medium for the ovarian cancer organoid of any one of claims 1 to 7;
(2) Acquiring ovarian cancer primary cells from an ovarian cancer tissue sample, and mixing the suspension of the acquired ovarian cancer primary cells with matrigel;
(3) Adding the culture medium of the ovarian cancer organoid obtained in the step (1) into the mixture of the ovarian cancer primary cells obtained in the step (2) and the matrigel for culturing.
9. A method for evaluating or screening a drug for treating ovarian cancer, comprising the steps of:
(1) Culturing an ovarian cancer organoid using the method of culturing an ovarian cancer organoid according to claim 8;
(2) Selecting a medicine to be detected and diluting according to a required concentration gradient;
(3) Adding the diluted medicine to the organoid cultured in (1);
(4) Organoid size or organoid viability assays are performed.
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| CN109652376B (en) * | 2019-01-08 | 2021-10-15 | 创芯国际生物科技(广州)有限公司 | Culture medium for 3D culture of ovarian cancer tissues |
| CN113528444B (en) * | 2020-04-15 | 2023-05-23 | 合肥中科普瑞昇生物医药科技有限公司 | Culture medium for esophageal squamous carcinoma epithelial cells, culture method and application thereof |
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