JP2021065129A - Method for producing gynecologic cancer organoid, gynecologic cancer organoid, culture medium for the production of gynecologic cancer organoid, and drug action evaluation method - Google Patents

Abstract

To provide a technique that produces a gynecologic cancer organoid that has excellent proliferative properties and is functionally similar to the original tissue.SOLUTION: A method for producing a gynecologic cancer organoid includes culturing gynecologic cancer-derived cell aggregate in a culture medium containing 5 μg/mL-50 μg/mL of fatty acids.SELECTED DRAWING: None

Description

The present invention relates to a method for producing a gynecologic cancer organoid, a gynecologic cancer organoid, a medium for producing a gynecologic cancer organoid, and a method for evaluating drug efficacy.

Ovarian cancer is extremely difficult to detect because it has no subjective symptoms and the ovary is an intra-abdominal organ, and when ovarian cancer is found, it is often found in an advanced state. Therefore, the 5-year survival rate of ovarian cancer patients is about 40 to 50%, and the survival rate of Stage III and Stage IV has not been improved for the past 40 years.

The main treatment for ovarian cancer patients is to first remove the cancer by excisional surgery and then treat with standard chemotherapy such as TC therapy. However, because many ovarian cancers are tumor heterogeneous, standard chemotherapy alone cannot completely eliminate the cancer, and as a result, it often recurs. Therefore, in the treatment of ovarian cancer patients, it is important to establish treatment after the second line.

As a second-line treatment, a method of searching for and treating a drug that responds by genome analysis of patient-derived ovarian cancer tissue is being investigated. However, it is known that the probability of finding a gene mutation in genome analysis is about 50%, and the probability of finding a drug expected to be effective is about 10%. Drug response is expected to be associated not only with gene mutations, but also with drug metabolism and other gene expression in cancer tissues. Therefore, in addition to genome analysis, the probability of finding a drug that may be effective can be increased by examining the drug susceptibility of the cancer tissue using the patient's ovarian tissue or a tissue functionally close to the tissue. it is conceivable that. In other words, in order to establish a second-line treatment, it is necessary to investigate drug metabolism in cancer tissues in addition to genome analysis.

Organoid cultures have the potential to produce cultures that have a structure that is anatomically very close to the tissue. In particular, organoids obtained from patient tissues may be able to have functions close to those tissues. Therefore, if an ovarian cancer organoid that is functionally close to the ovarian cancer tissue can be produced by culturing from the patient-derived ovarian cancer tissue with good proliferation, the ovarian cancer organoid can be used to establish a second-line treatment. It is considered possible to investigate the necessary drug susceptibility of cancer tissues. Non-Patent Document 1 is an example of ovarian cancer organoid culture from patient-derived ovarian cancer tissue.

Kopper O., et al., An organoid platform for ovarian cancer captures intra- and interpatient heterogeneity, Nature Medicine, 25 (5), 838-849, 2019.

INDUSTRIAL APPLICABILITY The present invention relates to a method for producing a gynecologic cancer organoid having excellent proliferative properties and functionally close to the tissue of origin, a medium for producing gynecologic cancer organoids, and a gynecologic cancer organoid, and a medicinal effect of a test substance using the gynecologic cancer organoid. The subject is to provide an evaluation method.

The present invention includes the following embodiments.
[1] A method for producing a gynecological cancer organoid, which comprises a step of culturing a cell aggregate derived from gynecological cancer in a medium containing a fatty acid of 5 μg / mL to 50 μg / mL.
[2] The method for producing a gynecologic cancer organoid according to [1], wherein the fatty acid is an unsaturated fatty acid.
[3] The method for producing a gynecologic cancer organoid according to [2], wherein the unsaturated fatty acid contains at least one selected from linoleic acid, linolenic acid and oleic acid.
[4] Production of the gynecological cancer organoid according to [3], wherein the unsaturated fatty acid contains 0.1 μg / mL to 20 μg / mL linoleic acid and 0.1 μg / mL to 20 μg / mL linolenic acid. Method.
[5] The method for producing a gynecologic cancer organoid according to [4], wherein the content of the linolenic acid is 0.5 to 20 times the content of the linoleic acid contained in the medium.
[6] The method for producing a gynecologic cancer organoid according to any one of [1] to [5], wherein the medium further contains a growth factor.
[7] The method for producing a gynecologic cancer organoid according to [6], wherein the growth factor contains insulin-like growth factor 1 and fibroblast growth factor 2.
[8] The method for producing a gynecologic cancer organoid according to any one of [1] to [7], wherein the medium further contains a bone morphogenetic protein inhibitor.
[9] The method for producing a gynecologic cancer organoid according to [8], wherein the bone morphogenetic protein inhibitor is noggin.
[10] The method for producing a gynecologic cancer organoid according to any one of [1] to [9], wherein the medium further contains a Wnt signal enhancer.
[11] The method for producing a gynecologic cancer organoid according to [10], wherein the Wnt signal enhancer comprises at least one selected from Wnt family members and R-sponge family members.
[12] The method for producing a gynecologic cancer organoid according to any one of [1] to [11], wherein the medium further contains a transforming factor β inhibitor.
[13] The method for producing a gynecologic cancer organoid according to any one of [1] to [12], wherein the medium further contains a Rho kinase inhibitor.
[14] The method for producing a gynecologic cancer organoid according to any one of [1] to [13], wherein the medium further contains a neurobiological supplement.
[15] The method for producing a gynecologic cancer organoid according to any one of [1] to [14], wherein the culture is a suspension culture.
[16] The method for producing a gynecologic cancer organoid according to any one of claims 1 to 15, wherein the gynecologic cancer is ovarian cancer.
[17] The method for producing a gynecologic cancer organoid according to [16], wherein the ovarian cancer is of human origin.
[18] The method for producing a gynecologic cancer organoid according to [16] or [17], wherein the ovarian cancer is an epithelial tumor.
[19] The method for producing a gynecologic cancer organoid according to [18], wherein the epithelial tumor is a serous adenocarcinoma, a clear cell adenocarcinoma, and an endometrial adenocarcinoma.
[20] A gynecologic cancer organoid produced by the method for producing a gynecologic cancer organoid according to any one of [1] to [19].
[21] A method for evaluating the efficacy of a test substance, which comprises a step of contacting the gynecological cancer organoid according to [20] with a test substance, and a step of measuring the effect of the test substance on the gynecological cancer organoid.
[22] A medium for producing gynecologic cancer organoids, which comprises 5 μg / mL to 50 μg / mL of fatty acid.

According to the method for producing a gynecologic cancer organoid according to one embodiment of the present invention, it is possible to produce a gynecologic cancer organoid having excellent proliferative properties and functionally close to the tissue of origin.

1 (a) to 1 (h) are micrographs of ovarian cancer organoids of Examples 1B to 3B and Comparative Examples 1B to 7B. 2 (a) to 2 (d) are micrographs of ovarian cancer organoids of Examples 4B to 6B and Comparative Example 8B. 3 (a) to 3 (d) are micrographs of ovarian cancer organoids of Examples 4C to 6C and Comparative Example 8C. 4 (a) to 4 (d) are graphs showing the results of Examples 1E to 4E. 5 (a) to 5 (c) are graphs showing the results of Examples 5E to 7E.

Hereinafter, the present invention will be described in more detail with reference to embodiments, but the present invention is not limited to the following embodiments.

In the present specification, the notation representing a numerical range such as "A to B" is synonymous with "A or more and B or less", and A and B are included in the numerical range.

Unless otherwise specified, each substance exemplified in the present specification, for example, a substance contained in a medium or a substance used in each step, may be used alone or in combination of two or more. it can.

In the present specification, "medium containing substance X" and "in the presence of substance X" mean a medium to which an exogenous substance X is added, a medium containing an exogenous substance X, or a foreign substance. It means the presence of substance X. That is, when a cell or tissue present in the medium expresses, secretes or produces the substance X endogenously, the endogenous substance X is distinguished from the exogenous substance X and is an exogenous substance. A medium containing no X does not fall under the category of "medium containing a substance X" even if it contains an intrinsic substance X.

[Manufacturing method of gynecologic cancer organoid]
The method for producing a gynecological cancer organoid of the present embodiment includes a step of culturing a cell aggregate derived from gynecological cancer in a medium containing 5 μg / mL to 50 μg / mL of fatty acid. The culture method is usually suspension culture, and when suspension culture is performed, stirring can be performed. The medium is the medium for producing gynecologic cancer organoids of the present embodiment.

The cell aggregate refers to an aggregate in which cells adhere to each other to form a cell population of 100 μm to 1000 μm. The cell agglutinin can usually be obtained by subjecting a gynecologic cancer sample (hereinafter, also referred to as “specimen”) to fragmentation, dispersion treatment, hemolysis treatment, or the like, if necessary.

Suspension culture is a culturing method in which cell aggregates are suspended in a medium without being adhered to the culture surface of an incubator used for culturing. When performing suspension culture, suspension culture may be performed with the cell aggregates embedded in the extracellular matrix, or suspension culture may be performed in a medium mixed with the extracellular matrix.

Examples of the incubator used for culturing include flasks, tissue culture flasks, dishes, petri dishes, tissue culture dishes, multi-dish, microplates, microwell plates, multi-plates, multi-well plates, chamber slides, petri dishes, and petri dishes. Examples include tubes, trays, culture bags, and roller bottles.

The culture surface of the incubator is preferably cell non-adhesive. Examples of the incubator having a cell non-adhesive culture surface include those subjected to cell non-adhesive treatment such as MPC polymer and those having a shape processed into irregularities.

The culture temperature is usually 20 ° C to 50 ° C, preferably 30 ° C to 40 ° C. _{The CO 2} concentration in the container during culturing is usually 1% by volume to 10% by volume. Within the above range, gynecologic cancer organoids can be efficiently produced.

The culture period is usually 6 days or more, preferably 6 to 14 days. During culturing, the components in the medium may be inactivated and disappear, so the medium is usually changed every 3 to 4 days.

Gynecologic cancers include Muller's duct cancer, ovarian cancer, peritoneal cancer, fallopian tube cancer, serous cancer of the uterine body, and the like. Examples of ovarian cancer include epithelial tumors, embryonic cell tumors, and interstitial sex cord tumors. Epithelial tumors include choriocarcinomas such as hyperatypical serous adenocarcinoma and low atypical serous adenocarcinoma, clear cell adenocarcinoma, endometrioid adenocarcinoma, mucinous adenocarcinoma, transitional epithelial tumor, and squamous epithelial tumor. Examples include mixed epithelial tumors, undifferentiated cancers, and adenocarcinomas of the ovarian network. Examples of embryonic cell tumors include disgernoma, yolk sac tumor, embryonic cancer, non-gestational fibrous carcinoma, and malformation cancer, and mixed embryonic cell tumors thereof.

Examples of gynecologic cancers include benign tumors obtained from primary tumors, borderline malignant tumors, malignant tumors, and malignant tumors obtained from metastatic cancers.

Gynecologic cancers may be of human or non-human animal origin. Non-human animals include, for example, mice, rats, dogs, cats, horses, monkeys, cows, sheep, pigs, goats, rabbits, hamsters, and guinea pigs.

Specimens can be obtained by removal by surgery or the like, or by collecting with an injection needle or an endoscope. Specimens are usually stored or transported at -5 ° C to 23 ° C by ice cooling. In addition, when storing or transporting the sample, it is placed in a cell preservation solution in order to increase the survival rate of the cells of the sample. Examples of commercially available cell preservation solutions include Dulbecco MEM such as DMEM / F-12, Eagle MEM, RPMI, Ham's F12, to which 1% GlutaMAX and 1% Pen Strip (Thermo Fisher Scientific) are added. Examples include Alpha MEM and Dulbecco modified by Iskov (all trade names above). In addition, when the specimen is removed or collected, or when the specimen is stored or transported, some tissues of the specimen may be necrotic. Therefore, the necrotic specimen should be a medical blade such as an ophthalmic blade. Can be removed using.

The sample is usually washed with a washing solution. Examples of the cleaning solution include Hanks' NAlanced Salt Solution (abbreviated as "HBSS", manufactured by Sigma Aldrich), acetate buffer (acetate + sodium acetate), phosphate buffer (phosphate + sodium phosphate, hereinafter "PHS". , Citrate buffer (citrate + sodium citrate), borate buffer, tartrate buffer, Tris buffer, and phosphate buffered saline (hereinafter, also referred to as "PBS"). Liquid is mentioned. Among these, HBSS is preferable. The number of times the sample is washed is usually 1 to 3 times.

If the sample is large and uneven in size and shape, the sample is usually fragmented. Slicing can usually be done with a knife, scissors, a cutter or the like. The size and shape of the sample after fragmentation are usually 1 mm to 5 mm square, more preferably 1 mm to 2 mm square, and the size and shape are preferably uniform.

Specimens or fragmented specimens can be dispersed. Dispersion means that cells are separated into 1 to 100 cell populations, preferably 5 to 50 cell populations by dispersion treatment such as enzyme treatment or physical treatment. Examples of the dispersion treatment include mechanical dispersion treatment, cell dispersion liquid treatment, and cell protective agent addition treatment. These processes can be combined. Among these, cell dispersion treatment is preferable.

Examples of the cell dispersion used for the cell dispersion treatment include collagenase, trypsin, papain, hyaluronidase, and C.I. Examples thereof include solutions containing any of enzymes such as historyticum neutral protease, thermolysin, and dispase; chelating agents such as ethylenediaminetetraacetic acid. The cell dispersion is usually an isotonic salt solution buffered at a physiologically acceptable pH, usually 6.0-8.0, preferably 7.2-7.6. Examples of the salt solution medium include PBS, HBSS, and the like. The treatment conditions for the cell dispersion treatment are usually 20 ° C. to 40 ° C., preferably 25 ° C. to 39 ° C., usually 1 minute to 180 minutes, preferably 30 minutes to 150 minutes.

After dispersion, hemolysis treatment can be performed. The hemolytic treatment is usually performed using a hemolytic agent. Examples of the hemolytic agent include surfactants such as ammonium chloride, sodium chloride, ammonium oxalate, saponin, and sodium lauryl sulfate, and other commercially available hemolytic agents such as Immunoprep (Beckman Coulter product name) and Immunorise (Beckman Coulter product). Name), BD FACS® (registered trademark) Lysing Solution (product name of Beckman Dickinson), and ASK Lysing Buffer (product name of Thermo Fisher Scientific).

The culture is preferably a primary culture. The gynecologic cancer organoid produced by the primary culture can exhibit the same behavior as that of the living body because it has not been long since the sample was collected from the living body.

In the 1-passage culture, the cell aggregates in the primary culture are taken out from the medium, placed in a cell preservation solution to stop the growth, and then the cell aggregates are dispersed as necessary, and the cell aggregates are dispersed in the same manner as in the primary culture. Is carried out by culturing in a medium. Similarly, the 2-passage culture is carried out by removing the cell aggregates in the 1-passage culture from the medium and performing the same operation as in the 1-passage culture. By repeating such an operation, subculture can be performed. Examples of the cell preservation solution include those described above.

[Medium for producing gynecologic cancer organoids]
The medium for producing gynecologic cancer organoids (hereinafter, also referred to as “medium”) of the present embodiment contains 5 μg / mL to 50 μg / mL of fatty acids. The medium is usually prepared by adding fatty acids to the basal medium. The medium can further contain extracellular matrix, growth factors, bone morphogenetic protein inhibitors, Wnt signal enhancers, Wnt signal enhancers, transformants-β inhibitors, Rho kinase inhibitors, and other components. ..

《Basic medium》
Examples of the basal medium include a medium used for culturing animal cells. Examples of the medium used for culturing animal cells include Eagle's Basic Medium (BME), BGJb Medium, CMRL-1066, Glasgow's MEM (GMEM), Improved MEM Zinc Option, and Iskov Modified Dalveco Medium (IMDM), 199 Medium. Examples include Medium 199, Eagle's MEM medium, Eagle's minimum essential medium α-modified (α-MEM), Dalveco's modified Eagle's medium (DMEM), RPMI 1640 medium, and Advanced DMEM / F-12 (all product names). .. One of these media may be used alone, or a plurality of types may be mixed and used.

"fatty acid"
The medium of the present embodiment contains fatty acids. Examples of fatty acids include saturated fatty acids such as lauric acid, myristic acid, palmitic acid and stearic acid, and unsaturated fatty acids such as linoleic acid, linolenic acid, and oleic acid, eicosapentaenoic acid, and docosahexaenoic acid. Among these, the fatty acid is preferably an unsaturated fatty acid, and among the unsaturated fatty acids, at least one selected from linoleic acid, linolenic acid, and oleic acid is more preferable, and linoleic acid and linolenic acid are used in combination. Is more preferable.

The content of fatty acids contained in the medium has a final concentration of 5 μg / mL to 50 μg / mL, preferably 6 μg / mL to 40 μg / mL, more preferably 7 μg / mL to 30 μg / mL, and even more preferably 8 μg / mL. It is mL to 25 μg / mL. Within the above range, gynecologic cancer organoids having excellent proliferative properties and functionally close to the tissue of origin can be produced. The final concentration indicates the concentration of the specific component contained in the medium as a result of adding the specific component to the medium.

When linoleic acid and linolenic acid are contained as fatty acids, the content of linoleic acid contained in the medium is 5 μg / mL to 20 μg / mL, preferably 5 μg / mL to 15 μg / mL, and the content of linolenic acid. Is 5 μg / mL to 20 μg / mL, preferably 5 μg / mL to 15 μg / mL. Within the above range, a gynecologic cancer organoid having excellent proliferative potential and a high expression level of a tumor stem cell marker can be produced.

When linoleic acid and linolenic acid are contained as fatty acids, the content of the linolenic acid is 0.5 to 20 times, more preferably 0.6 times to the content of the linolenic acid contained in the medium. It is preferably 15 times, more preferably 0.8 to 13 times.

《Extracellular matrix》
The medium preferably further comprises an extracellular matrix. Examples of the extracellular matrix include components contained in the basement membrane and glycoproteins present in the intercellular spaces. Examples of the components contained in the basement membrane include type IV collagen, laminin, heparan sulfate proteoglycan, and entactin. As the extracellular matrix, a commercially available product containing the extracellular matrix can be used. Examples of glycoproteins present in the intercellular spaces include collagen, laminin, entactin, fibronectin, heparin sulfate and the like. Examples of commercially available products containing extracellular matrix include Matrigel (Corning Inc. product name) and Human Laminin (Sigma Inc. product name).

Matrigel contains a basement membrane component derived from Englbreth Holm Swarm mouse sarcoma. The main components of Matrigel are type IV collagen, laminin, heparan sulfate proteoglycan, and entactin, but in addition to these, epidermal growth factor (hereinafter, also referred to as "EGF") and nerve growth factor (hereinafter, also referred to as "NGF"). ), Platelet-derived growth factor (hereinafter, also referred to as "PDGF"), (hereinafter, insulin-like growth factor 1 "IGF-1"), and transforming factor β (hereinafter, also referred to as "TGF-β"). Various growth factors such as are included. Matrigel is also available in grades with low concentrations of various growth factors, with EGF less than 0.5 ng / mL, NGF less than 0.2 ng / mL, PDGF less than 5 pg / mL, and IGF-1 less than 5 ng / mL. , TGF-β is less than 1.7 ng / mL. As the matrigel, it is preferable to use a grade having a low content ratio of various growth factors.

The extracellular matrix in the medium can be used to embed cell aggregates, or can be used in a state of being mixed with a medium component other than the extracellular matrix.

In the medium mixed with the extracellular matrix, the volume fraction of the extracellular matrix is usually 2% by volume or more, preferably 2% by volume to 10% by volume, based on the volume of components other than the extracellular matrix in the medium. Can be prepared by mixing the above amounts. Examples of the mixing method include a pipetting method on an ice bath. Mixing means a state in which the extracellular matrix is not visually observed in the medium.

《Growth factor》
The medium preferably further contains growth factors. Examples of growth factors include insulin-like growth factor (hereinafter, also referred to as “IGF”), epidermal growth factor, fibroblast growth factor (hereinafter, also referred to as “FGF”), epiregulin (hereinafter, also referred to as “EREG”), and the like. Can be mentioned. Among these, as the growth factor, it is preferable to have at least one selected from IGF and FGF.

Examples of IGF include IGF1 and IGF2, and among these, it is preferable that IGF1 is contained at least. Examples of FGF include FGF1 to FGF23, and among these, it is preferable that FGF2 is contained at least.

The final concentration of the growth factor contained in the medium is usually 5 ng / mL to 10 μg / mL, preferably 10 ng / mL to 1 μg / mL, and more preferably 50 ng / mL to 500 ng / mL.

When at least IGF1 and FGF2 are contained as growth factors contained in the medium, the content ratio of IGF1 contained in the medium has a final concentration of 5 ng / mL to 10 μg / mL, preferably 10 ng / mL to 1 μg / mL. It is mL, more preferably 50 ng / mL to 500 ng / mL, and the content ratio of FGF2 contained in the medium is such that the final concentration is 5 ng / mL to 10 μg / mL, preferably 10 ng / mL to 1 μg / mL, more preferably. Is 50 ng / mL to 500 ng / mL.

《Bone morphogenetic protein inhibitor》
The medium preferably contains a bone morphogenetic protein inhibitor (hereinafter, also referred to as “BMP inhibitor”). By including a BMP inhibitor in the medium, the loss of stem cells can be suppressed.

BMP inhibitors include chordin-like proteins including nogin, gremlin, chordin, and chordin domains; follistatin, and follistatin-related proteins, including follistatin domains; DAN, and DAN-like proteins, including DAN cysteine knot domains; sclerostin / SOST; and α-2 macroglobulin are mentioned, of which nogin is preferred.

The content ratio of the BMP inhibitor contained in the medium is such that the final concentration is 10 ng / mL to 400 ng / mL, preferably 20 ng / mL to 300 ng / mL, and more preferably 50 ng / mL to 200 ng / mL.

<< Wnt signal enhancer >>
The medium preferably further contains a Wnt signal enhancer. Examples of Wnt signal enhancers include R-spondins such as Wnt family members, R-spondin 1, R-spondin 2, R-spondin 3, and R-spondin 4; norin, and GSK-inhibitors. Among these, Wnt family members and R-spondin are preferable.

Examples of Wnt family members include Wnt1, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a, Wnt9b, Wnt10a, Wnt10b, Wnt11 and Wnt16.

The Wnt family member is preferably Wnt3a or Wnt4, more preferably Wnt3a. Wnt3a is more preferably a complex with afamin in order to improve stability.

When a complex of Wnt3a and afamin is contained as a Wnt signal enhancer, the content ratio of the complex of Wnt3a and afamin contained in the medium for producing organoids for gynecologic cancer has a final concentration of 50 ng / mL to 10 μg / mL. , Preferably 100 ng / mL to 5 μg / mL, and more preferably 200 ng / mL to 1 μg / mL.

When R-spondin is contained as a Wnt signal enhancer, the content ratio of R-spondin contained in the medium for producing organoids for gynecologic cancer has a final concentration of 10 ng / mL to 10 μg / mL, preferably 30 ng / mL to 1 μg. / ML, more preferably 50 ng / mL to 200 ng / mL.

<< Transforming factor-β inhibitor >>
The medium preferably further contains a transforming factor-β inhibitor (hereinafter, also referred to as “TGF-β inhibitor”). A TGF-β inhibitor is one or more inhibitors that directly or indirectly negatively regulate TGF-β signaling.

A TGF-β inhibitor is a component that binds to serine / threonine protein kinases such as ALK5, ALK4, TGF-β receptor kinase 1 and ALK7 and reduces their activity.

Examples of the TGF-β inhibitor include A83-01 (CAS number: 909910-43-6), SB-431542 (CAS number: 301836-41-9), SB-505124 (CAS number: 694433-59-5), SB-525334 (CAS number: 356559-20-1), SD-208 (CAS number: 627536-09-8), LY-36497 (CAS number: 396129-53-6), and SJN-2511 (CAS number:: 446859-33-2) and the like. Of these, A83-01 is preferable.

The content ratio of the TGF-β inhibitor contained in the medium is such that the final concentration is 100 nM to 10 μM, preferably 200 nM to 5 μM, and more preferably 500 nM to 2 μM.

<< Rho-kinase inhibitor >>
The medium preferably further contains a Rho-kinase inhibitor (hereinafter, also referred to as "ROCK inhibitor"). Examples of the ROCK inhibitor include Y-27632 (CAS number: 129830-38-2), Derivative / HA1077 (CAS number: 103745-39-7), H-1152 (CAS number: 871543-07-6), and the like. And Wf-536 (CAS Registry Number: 539857-64-2), and derivatives thereof.

The content ratio of the ROCK inhibitor contained in the medium is such that the final concentration is 1 μM to 50 μM, preferably 3 μM to 20 μM, and more preferably 5 μM to 10 μM.

<< Other ingredients >>
The medium can further contain, as other components, serum, serum substitutes, neurobiological supplements, medium supplements, serum-derived proteins such as insulin and albumin, and the like.

Serum alternatives include, for example, albumin, amino acids, transferrin, insulin, collagen precursors, trace elements, 2-mercaptoethanol, and 3'thiolglycerol, and equivalents thereof.

Commercially available serum alternatives include, for example, Knockout Serum Replacement (Thermo Fisher Scientific Product Name), Chemically-defined Lipid Concentrated Fatty Acid Concentrate (Thermo Fisher Scientific Product Name), and GlutaMAX (Thermo Fisher Scientific). Fic's product name).

Neurobiological supplements include biotin, cholesterol, linoleic acid, linolenic acid, progesterone, putresin, retinol, retinyl acetate, sodium selenite, triiodothyronine (T3), DL-α-tocopherol (vitamin E), and albumin. , B27 supplements containing insulin and transferase (Thermofisher product name); and N2 supplements containing human transferase, bovine insulin, progesterone, putresin, and sodium selenite.

[Gynecologic Cancer Organoid]
The gynecologic cancer organoid of the present embodiment is a gynecologic cancer organoid produced by the method for producing a gynecologic cancer organoid of the present embodiment. The gynecologic cancer organoid of the present embodiment has a structure anatomically very close to that of a gynecologic cancer sample. Further, since the gynecologic cancer organoid of the present embodiment depends on the individuality of the gynecologic cancer sample, it is not possible to specify the expression pattern of a gene marker, a protein marker, or the like.

The growth rate of gynecological cancer organoids can be evaluated by, for example, a method of visually observing the number of viable cells together with a control, or a method of measuring the number of viable cells by image analysis after imaging with a CCD camera or the like.

When the gynecologic cancer is ovarian cancer and the type of ovarian cancer is epithelial tumor, the expression level of mRNA of Lgr5 gene, which is a stem cell marker of epithelial tumor, can be measured by real-time qPCR method. In measuring the expression level, the expression level is corrected by the endogenous control gene. When measuring the expression level of the mRNA of the Lgr5 gene, examples of the endogenous control gene include the GAPDH gene and the ACTB gene.

[Method for evaluating the efficacy of the test substance]
The method for evaluating the efficacy of the test substance of the present embodiment is a step of bringing the gynecological cancer organoid into contact with the test substance (hereinafter, also referred to as “step 1”), and a step of measuring the effect of the test substance on the gynecological cancer organoid. (Hereinafter, also referred to as "step 2").

<< Process 1 >>
In step 1, the gynecologic cancer organoid is brought into contact with the test substance. Examples of the test substance include all known agents including various anticancer agents. In addition, radiation for radiotherapy can also be included as a drug.

<< Process 2 >>
In step 2, the effect of the test substance on the gynecologic cancer organoid is measured. Examples of the method for measuring the effect of the test substance on the gynecologic cancer organoid include a method for measuring the amount of ATP contained in cells with a detection reagent. Examples of the ATP detection reagent include commercially available luminescent reagents (trade name "CellTiter-Glo", catalog number "G7570", Promega) and the like. Other ATP detection reagents include a method of colorimetrically measuring the number of cells as the amount of protein by staining with a protein-binding dye. Examples of the protein-binding dye include sulforhodamine B.

Examples of the method for measuring the effect of other test substances on the gynecological cancer organoid include a method for measuring SD (Succinyl dehidrogenase) activity, a method for measuring the number of surviving cells by an MTT assay, an MTS assay and the like.

By the method for evaluating the efficacy of the test substance of the present embodiment, the type of the test substance to which the gynecologic cancer organod is sensitive can be specified. In addition, the presence or absence of radiation sensitivity of gynecologic cancer organoids can be evaluated. Then, the obtained evaluation result can be applied to the treatment of the patient from which the gynecologic cancer tissue is derived. Therefore, the method for evaluating the efficacy of the test substance of the present embodiment is useful for individually confirming an effective treatment method for each patient.

The method for evaluating the efficacy of a test substance according to the present embodiment can produce a gynecologic cancer organoid from a small amount of sample and make an efficient prediction, so that the burden on the patient can be reduced. Furthermore, it is possible to elucidate the unknown relationship between genes and drug sensitivity or radiosensitivity.

In recent years, from the viewpoint of side effects and medical economics, there is an increasing need to preliminarily test the drug sensitivity or radiosensitivity of patients and select patients for whom the drug is effective. For example, since a target molecule and its intracellular signal have been clarified for a molecular-targeted drug, it may be possible to determine the efficacy of the drug by searching for a mutation in the target gene in molecular biology.

Such a gene is not particularly limited, and may be a gene peculiar to cancer, or may reflect the constitution or metabolism of animals including humans. For example, a KRAS gene or a BRAF gene can be mentioned as a typical one whose relationship with drug susceptibility has been clarified.

Mutations in the oncogenes KRAS or BRAF have been shown to have the potential to be used to predict the effects of cetuximab, an antibody drug against epidermal growth factor receptor (EGFR), on colorectal cancer. In patients with the mutation, the effect of cetuximab is inadequate.

However, there is a technical limit in cases where the judgment must be made from a limited amount of biopsy material and there is no surgical indication. Especially in the case of cancer tissue having a small amount of cancer cell components, it is very difficult to detect an abnormality.

According to the above-mentioned method for producing a gynecologic cancer organoid, a purified gynecologic cancer organoid can be produced. By culturing a small amount of sample and purifying and amplifying cancer cells, it is possible to accurately analyze genes such as KRAS gene or BRAF gene.

As the gene analysis, detection of polymorphisms such as UGT1A1 gene polymorphism can be used. The UGT1A1 gene is also known to have low or low susceptibility to anticancer drugs due to polymorphism, and by obtaining such information in advance, administration of a drug that induces only side effects can be avoided.

Such an evaluation can be, for example, detecting the presence or absence of a mutation in a gene. Here, the mutation includes all kinds of diversity such as deletion as well as base change. Detection of gene mutations can be performed by any known method, such as directly sequencing the bases contained in the gene or evaluating the restriction enzyme cleavage site.

The step of evaluating the gene may be to detect the gene expression level. The gene expression level can be measured by detecting the expression or expression level of mRNA, which is a transcript of the gene, or the presence or abundance of a protein or protein fragment, which is a translation product of the gene.

Transcripts of genes can be detected or measured by known methods such as Northern blotting, RT-PCR, in situ hybridization, and DNA microarray methods that specifically detect the expression of a specific gene.

As a method of gene evaluation, it may be carried out by culturing in a hypoxic state and a normal oxygen state, and comparing the gene expression level in the culture under the hypoxic state and the normal oxygen state. Examples of the gene suitable for such an evaluation method include, but are not limited to, the VEGF gene and the like. The information obtained from the VEGF gene relates to the clinical application of angiogenesis inhibitors to therapeutic agents for colorectal cancer.

For example, bevacizumab is a humanized monoclonal antibody against vascular endothelial growth factor (VEGF). VEGF promotes cell division of vascular endothelial cells and is upregulated in various cancer cells. It is thought that VEGF promotes angiogenesis to increase the supply of nutrients and oxygen, and is involved in the growth and metastasis of cancer cells.

Bevacizumab exhibits anti-cancer activity by specifically binding to VEGF and inhibiting its biological activity. At present, there are no effective studies predicting the drug susceptibility of angiogenesis inhibitors. Although it may be important to assess the VEGF-producing ability of tumors, histopathological analysis of VEGF in previous studies does not reflect therapeutic effects.

Since the internal environment of the tumor is extremely heterogeneous and it is difficult to identify where angiogenesis is active, assessing VEGF production in the tumor as a whole may not necessarily lead to susceptibility prediction. There is.

Cancer tissues are known to be hypoxic, which is the most potent VEGF inducer. For example, in the drug efficacy evaluation method of the present embodiment, the "potential" of cancer cells can be evaluated by changing the culture conditions.

Specific examples of the present embodiment will be described below with reference to the embodiments, but the present embodiment is not limited thereto. All experiments were conducted based on the ethics research plan approved by the Keio University School of Medicine Ethics Committee.

[Examples 1A to 6A, and Comparative Examples 1A to 8A] Production of medium for gynecological cancer organoid production As a basal medium (product name "Advanced DMEM / F-12", manufactured by Thermo Fisher Scientific Co., Ltd.) , The components shown in Table 1 below were mixed to the final concentrations shown in Table 1 below to prepare two types of base media. These base media are referred to as "IFNRA medium" and "WIGNRA medium", respectively. The fatty acids shown in Table 2 below are further mixed with these base media to the final concentrations shown in Table 2 below to produce ovarian cancer organoids of Examples 1A to 6A and Comparative Examples 1A to 8A. A medium for production was produced.

Since the medium containing the B27 supplement having a final concentration of 2% by volume contains linoleic acid having a final concentration of 1 μg / mL and linolenic acid having a final concentration of 1 μg / mL, the final concentrations of linoleic acid and linolenic acid in Table 1 The concentration is a value including linoleic acid and linolenic acid contained in the B27 supplement.

[Preparation Example 1] Preparation of cell aggregates Ovarian cancer (lesion tissue name: 18-146) was collected from ovarian tumor patients as pathological specimens. The 18-146 histological type was clear cell adenocarcinoma. The ovarian cancer tissue was placed in a centrifuge tube in a cell preservation solution (product name "Advanced DMEM / F-12", manufactured by Thermo Fisher Scientific) under aseptic technique, and cooled on ice in the laboratory. Transported to. In the safety cabinet, the cell preservation solution used during transportation was removed, and 20 mL of ice-cooled washing solution (product name "HBSS, no calcium, no magnesium", manufactured by Thermo Fisher Scientific Co., Ltd.) was used for inversion and mixing. The ovarian cancer tissue was washed 3 times.

The ovarian cancer tissue was transferred to a 9 cm tissue culture dish (product name "cell culture petri dish 90Φ", manufactured by Sumitomo Bakelite Co., Ltd.). The culture dish was placed on ice and necrotic tissue was removed in the dish using ophthalmic scissors. The ovarian cancer tissue piece from which the necrotic tissue had been removed was cut into pieces of about 0.5 to 1 mm square using an ophthalmic scissors blade. Transfer the fragmented ovarian cancer tissue piece and 45 mL of washing solution to a 50 mL centrifuge tube and centrifuge (centrifugal force RCF: 400 × g, centrifugation time: 5 minutes), discard the supernatant, and add 45 mL of washing solution. , Overturned and mixed. The operation from centrifugation to inversion mixing was repeated twice.

10 mL of cell dispersion was added per 600 mg by mass of the fragmented ovarian cancer tissue pieces and mixed. Shake the container containing the fragmented ovarian cancer tissue pieces in a constant temperature bath (product name "Water Bath Personal-11", manufactured by TIETECH Co., Ltd.) at 37 ° C. and 160 rpm for 30 minutes to mesh the contents of the container. It was passed through a 100 μm cell strainer (manufactured by Falcon). Then, 40 mL cleaning solution is added and centrifuged (centrifugal force RCF: 400 × g, centrifugation time: 5 minutes), the supernatant is discarded, and then 40 mL cleaning solution is added and centrifuged (centrifugal force RCF: 400 × g). , Centrifugal time: 5 minutes), the supernatant was discarded.

The cell dispersion was prepared in 8.5 mL wash solution, 1.1 mL collagenase (final concentration 1 mg / mL), 0.55 mL Dispase II (final concentration 3.6 mg / mL), Y-27136 (final concentration 10 μM), And 100 U DNase I (Roche, 100 Units / 10 mL) were added.

A hemolytic agent (product name "ASK Lysing Buffer", manufactured by Thermo Fisher Scientific Co., Ltd.) was mixed with the cell aggregates after the dispersion treatment, and the cells were allowed to stand for 5 minutes for hemolysis treatment. Then, 40 mL washing solution is added and centrifuged (centrifugal force RCF: 400 × g, centrifugation time: 5 minutes), the supernatant is discarded, 40 mL washing solution is added, and the mixture is centrifuged (centrifugal force RCF: 400 × g, centrifugation time). : 5 minutes), discard the supernatant, add the cell preservation solution, centrifuge (centrifugal force RCF: 400 × g, centrifugation time: 5 minutes), discard the supernatant, and dispose of cell aggregates with a cell diameter of 11 μm or more. Obtained. The obtained cell aggregate is referred to as "18-146 cell aggregate".

[Preparation Example 2] Preparation of Cell Agglutination Example 1 of Preparation Example 1 except that ovarian cancer having lesion tissue name "19-010" was used instead of ovarian cancer having lesion tissue name "18-146" in Preparation Example 1. By the same operation as in the above, a cell aggregate having a cell diameter of 11 μm or more was obtained. The obtained cell aggregate is referred to as "19-010 cell aggregate". The ovarian cancer with the lesion tissue name "19-010" was a clear cell adenocarcinoma.

[Example 1B] Production of gynecological cancer organoid (0 passage primary culture) 25 μL of Matrigel (registered trademark, manufactured by Corning Inc.) in which 3000 18-146 cell aggregates produced in Preparation Example 1 were thawed under ice-cooling. ), And _{allowed to stand in a CO 2} incubator (37 ° C., 5% by volume CO ₂ ) for 10 minutes to gel Matrigel.

200 to 300 μL of the medium for producing ovarian cancer organoids shown in Table 3 was added to each well, and the cells were cultured in a CO ₂ incubator (37 ° C., 5% by volume CO ₂ ) for 14 days. 0 passage primary culture) was produced. In culturing, the medium was changed every 2 or 3 days after sowing.

FIG. 1 (i) is a photomicrograph of the ovarian cancer organoid of Example 1B. In FIG. 1 (i), "e / n-10" indicates that the final concentration of linoleic acid in the medium was 10 μg / mL and the final concentration of linolenic acid was 10 μg / mL.

[Examples 2B to 6B, and Comparative Examples 1B to 8B] Production of Gynecologic Cancer Organoids (0 Subculture Primary Culture) Except for using the cell aggregates and media for producing gynecologic cancer organoids shown in Table 3. Produced gynecological cancer organoids (0 passage primary culture) of Examples 2B to 6B and Comparative Examples 1B to 8B by the same operation as in Example 1B.

FIG. 1A is a photomicrograph of the ovarian cancer organoid of Comparative Example 1B. FIG. 1 (c) is a photomicrograph of the ovarian cancer organoid of Example 2B. FIG. 1 (f) is a photomicrograph of an ovarian cancer organoid of Example 3B. FIG. 1 (j) is a photomicrograph of the ovarian cancer organoid of Comparative Example 2B. FIG. 1 (d) is a photomicrograph of the ovarian cancer organoid of Comparative Example 3B. FIG. 1 (g) is a photomicrograph of the ovarian cancer organoid of Comparative Example 4B. FIG. 1B is a photomicrograph of the ovarian cancer organoid of Comparative Example 5B. FIG. 1 (e) is a photomicrograph of the ovarian cancer organoid of Comparative Example 6B. FIG. 1 (h) is a photomicrograph of the ovarian cancer organoid of Comparative Example 7B. In FIGS. 1A to 1H, "e" indicates linoleic acid, "n" indicates linolenic acid, and the numbers indicate the final concentration of linoleic acid or linolenic acid (μg / mL).

FIG. 2A is a photomicrograph of the ovarian cancer organoid of Comparative Example 8B. FIG. 2B is a photomicrograph of the ovarian cancer organoid of Comparative Example 4B. FIG. 2C is a photomicrograph of the ovarian cancer organoid of Comparative Example 5B. FIG. 2D is a photomicrograph of the ovarian cancer organoid of Comparative Example 6B. In FIGS. 2 (a) to 2 (d), "e" indicates linoleic acid, "n" indicates linolenic acid, "o" indicates oleic acid, and numbers indicate the end of linoleic acid, linolenic acid or oleic acid. Indicates the concentration (μg / mL).

[Example 4C] Production of gynecological cancer organoid (3 passage culture) In Example 4B, the medium was removed on the first day of culture, and 500 μL of a cell stripping agent per well (product name “TypeLE Express”, thermofisher). (Scientific) is added, and while breaking the matrix with a micropipette tip, the cells are treated in a 15 mL centrifuge tube at 37 ° C. for 10 minutes in a hot water bath, and the cell aggregates become 1 to 10 cell aggregates. Pipetting up to. Next, 10 mL of cell preservation solution (product name "Advanced DMEM / F-12", manufactured by Thermo Fisher Scientific Co., Ltd.) was added, and the mixture was centrifuged (centrifugal force RCF: 400 × g, centrifugation time: 5 minutes). I abandoned Qing.

Then, the cell aggregate was suspended in 200 μL of cell preservation solution (product name “Advanced DMEM / F-12”, manufactured by Thermo Fisher Scientific Co., Ltd.). 3,000 ovarian cancer organoids were seeded in a 48-well plate with 25 μL of Matrigel (registered trademark, manufactured by Corning Inc.) and _{allowed to stand in a CO 2} incubator (37 ° C., 5% by volume CO ₂ ) for 10 minutes. Was gelled. 200 μL to 300 μL of the medium for producing ovarian cancer organoid of Example 4A was added to each well, and the cells were cultured in a CO ₂ incubator (37 ° C., 5% by volume CO ₂ ) for 14 days. The ovarian cancer organoid obtained by culturing for 14 days was used as the first passage, and the same operation as described above was subsequently repeated to produce the third passage ovarian cancer organoid of Example 4C. Hereinafter, the third-generation ovarian cancer organoid is also referred to as "ovarian cancer organoid (third-generation primary culture)".

FIG. 3B is a photomicrograph of the ovarian cancer organoid of Example 4C. In FIG. 3 (b), "e / n-10" indicates that the final concentration of linoleic acid in the medium was 10 μg / mL and the final concentration of linolenic acid was 10 μg / mL.

[Examples 5C to 6C, and Comparative Example 8C] Production of gynecological cancer organoids (three subcultures) Except for using the 0-passage primary culture gynecological organoids and the medium for gynecological cancer organoid production shown in Table 4. Produced gynecological cancer organoids (three subcultures) of Examples 5C to 6C and Comparative Example 8C by the same operation as in Example 4C.

FIG. 3A is a photomicrograph of the ovarian cancer organoid of Comparative Example 8C. FIG. 3C is a photomicrograph of the ovarian cancer organoid of Example 5C. FIG. 3D is a photomicrograph of the ovarian cancer organoid of Example 6C. In FIGS. 3 (a), (c) and (d), "n" indicates linolenic acid, "o" indicates oleic acid, and the numbers indicate the final concentration of linolenic acid or oleic acid (μg / mL). ..

《Proliferative》
The proliferation of gynecologic cancer organoids was observed under a microscope and evaluated according to the following criteria. The evaluation results are shown in Table 2. In the judgment criteria, the "criteria" of Examples 1B to 3B and Comparative Examples 2B to 7B are Comparative Example 1B, and the "criteria" of Examples 4B to 6B are Comparative Example 8B and Examples 4C to. The "reference" of Example 6C is Comparative Example 8C.
(Criteria)
5: Very good compared to the standard 4: Good compared to the standard 3: Standard 2: Poor compared to the standard 1: Dead

<< Lgr5 expression level >>
The Lgr5 expression level of the gynecologic cancer organoid was evaluated as follows. MRNA was extracted from gynecologic cancer organoids using a commercially available kit (product name "FastLane Cell cDNA", manufactured by Kiagen), and the expression level of the mRNA of the Lgr5 gene, which is a stem cell marker for epithelial egg tumors, was measured in real time by qPCR. Measured by method. In addition, the expression level of GAPDH gene mRNA was used as the endogenous control gene. For the measurement, a commercially available primer (product name "TB Green Premix Ex Taq II", manufactured by Takara Bio Inc.) and a primer showing the base sequence in Table 5 below were used. The evaluation results are shown in Tables 3 and 4.

In Tables 3 and 4, the mRNA expression level of Lgr5 was corrected by the expression level of GAPDH, and for Examples 1B to 3B and Comparative Examples 2B to 7B, the mRNA of Lgr5 of Comparative Example 1B was used. The expression level was set to 1, the mRNA expression level of Lgr5 of Comparative Example 8B was set to 1 for Examples 4B to 6B, and the mRNA expression level of Lgr5 of Comparative Example 8C was set to 1 for Examples 4C to 6C. It is shown as a relative value when. Further, "ND" indicates undetectable.

[Example 1D to Example 7D] Production of gynecologic cancer organoid (3 passage culture) Two types of highly atypical serous adenocarcinoma (lesion tissue name: 18-) as pathological specimens from ovarian tumor patients shown in Table 6 below. 016 and lesion tissue name: 18-064), low atypical serous adenocarcinoma (lesion tissue name: 18-036), clear cell adenocarcinoma (lesion tissue name: 18-015), 3 types of endometrial adenocarcinoma The diameter was the same as in Preparation Example 1, Example 1B and Example 4C except that (lesion tissue name: 18-053, lesion tissue name: 18-055 and lesion tissue name: 18-065) were used. Gynecologic cancer organoids of about 100 μm from Example 1D to Example 7D were produced.

[Example 1E to Example 7E]
The gynecologic cancer organoids of Examples 1D-7D were exposed to anti-cancer agents. As anticancer agents, carboplatin, paclitaxel, olaparib, topotecan, gefitinib and lapatinib were used.

The ATP activity of gynecologic cancer organoids was measured 4 days after exposure to the anticancer drug. To measure ATP activity, a commercially available luminescent reagent (trade name "CellTiter-Glo", catalog number "G7570", Promega) was used to measure the issuance signal intensity of gynecological cancer organoids not exposed to anticancer agents. based on the issuance intensity of ATP activity, analysis software 50% inhibitory concentration (IC ₅₀₎ (product name "GraphPad prism", GraphPad software Inc.) non-linear regression analysis plotting the dose response curve for each drug using _{IC 50} values were calculated from.

4 (a) to 4 (d) and 5 (a) to 5 (c) are graphs showing a dose-response curve of each gynecologic cancer organoid. FIG. 4A is a graph showing the result of Example 4E, FIG. 4B is a graph showing the result of Example 1E, and FIG. 4C is a graph showing the result of Example 3E. 4 (d) is a graph showing the result of Example 2E, FIG. 5 (a) is a graph showing the result of Example 5E, and FIG. 5 (b) is a graph showing the result of Example 6E. Yes, FIG. 5 (c) is a graph showing the results of Example 7E. In FIGS. 4 (a) to 4 (d) and FIGS. 5 (a) to 5 (c), the vertical axis shows the ratio of ATP activity, and the horizontal axis shows the concentration of the exposed anticancer drug. In addition, the name of the lesion tissue used for the gynecologic cancer organoid is shown at the top of each graph.

According to the present invention, it is possible to provide a technique for producing a gynecological cancer organoid from a gynecological cancer tissue piece.

Claims (22)

A method for producing a gynecological cancer organoid, which comprises a step of culturing a cell aggregate derived from gynecological cancer in a medium containing 5 μg / mL to 50 μg / mL fatty acid. The method for producing a gynecologic cancer organoid according to claim 1, wherein the fatty acid is an unsaturated fatty acid. The method for producing a gynecological cancer organoid according to claim 2, wherein the unsaturated fatty acid contains at least one selected from linoleic acid, linolenic acid and oleic acid. The method for producing a gynecological cancer organoid according to claim 3, wherein the unsaturated fatty acid contains 0.1 μg / mL to 20 μg / mL linoleic acid and 0.1 μg / mL to 20 μg / mL linolenic acid. The method for producing a gynecologic cancer organoid according to claim 4, wherein the content of the linolenic acid is 0.5 to 20 times the content of the linoleic acid contained in the medium. The method for producing a gynecologic cancer organoid according to any one of claims 1 to 5, wherein the medium further contains a growth factor. The method for producing a gynecologic cancer organoid according to claim 6, wherein the growth factor contains insulin-like growth factor 1 and fibroblast growth factor 2. The method for producing a gynecological cancer organoid according to any one of claims 1 to 7, wherein the medium further contains a bone morphogenetic protein inhibitor. The method for producing a gynecologic cancer organoid according to claim 8, wherein the bone morphogenetic protein inhibitor is noggin. The method for producing a gynecologic cancer organoid according to any one of claims 1 to 9, wherein the medium further contains a Wnt signal enhancer. The method for producing a gynecologic cancer organoid according to claim 10, wherein the Wnt signal enhancer comprises at least one selected from Wnt family members and R-sponge family members. The method for producing a gynecologic cancer organoid according to any one of claims 1 to 11, wherein the medium further contains a transforming factor β inhibitor. The method for producing a gynecologic cancer organoid according to any one of claims 1 to 12, wherein the medium further contains a Rho kinase inhibitor. The method for producing a gynecologic cancer organoid according to any one of claims 1 to 13, wherein the medium further contains a neurobiological supplement. The method for producing a gynecologic cancer organoid according to any one of claims 1 to 14, wherein the culture is a suspension culture. The method for producing a gynecologic cancer organoid according to any one of claims 1 to 15, wherein the gynecologic cancer is ovarian cancer. The method for producing a gynecologic cancer organoid according to claim 16, wherein the ovarian cancer is of human origin. The method for producing a gynecologic cancer organoid according to claim 16 or 17, wherein the ovarian cancer is an epithelial tumor. The method for producing a gynecologic cancer organoid according to claim 18, wherein the epithelial tumor is a serous adenocarcinoma, a clear cell adenocarcinoma, and an endometrial adenocarcinoma. A gynecologic cancer organoid produced by the method for producing a gynecologic cancer organoid according to any one of claims 1 to 19. A method for evaluating the efficacy of a test substance, which comprises a step of contacting the gynecological cancer organoid according to claim 20 with a test substance, and a step of measuring the effect of the test substance on the gynecological cancer organoid. A medium for producing gynecologic cancer organoids containing 5 μg / mL to 50 μg / mL of fatty acids.

Images