JP2021145646A - Cell culture body and production method thereof - Google Patents

Abstract

To reflect an original feature of a cancer tissue derived from a patient such as a three dimensional organoid, and exhibit growth rate excellent relative to growth rate of the three-dimensional organoid.SOLUTION: A production method of a cell culture body comprises: a step for culturing a three-dimensional organoid on a medium for an animal cell; and a step for leaving cells adhered to a culture vessel, out of cells comprising migration function derived from the three-dimensional organoid, and removing a component other than the cells from the culture vessel.SELECTED DRAWING: Figure 1

Description

The present invention relates to a cell culture produced using a three-dimensional organoid and a method for producing the same.

The three-dimensional organoid culture method (Non-Patent Document 1: Sato et al., Nature, 2009) mixes epithelial cells isolated from various organs with Matrigel to enhance stem cell properties such as Wnt, Noggin, and R-spondin. It was developed as a method that can reproduce the three-dimensional epithelial tissue structure on a culture dish by culturing in a special medium containing a factor. In recent years, an organoid culture method using surgical specimens of patients such as human colon cancer and pancreatic cancer has been established, and structural similarity with the tissue immediately after excision and correlation with gene mutation have been shown (Non-Patent Document 2: Non-Patent Document 2: Wetering et al., Cell, 2015 and Non-Patent Document 3: Boj et al., Cell, 2015) are also expected to be useful tools for personalized medicine.

In addition, a technique for non-invasively culturing bladder cancer organoids using urine samples from dogs suffering from bladder cancer has been developed, and the produced organoids reproduce the characteristics of bladder cancer in vivo in three dimensions and immunize. It has been clarified that it can be applied to a tumor remodeling ability test in a defective mouse body and an anticancer drug susceptibility test for individual patients (Non-Patent Document 4: Elbadawy and Usui et al., Cancer Sci. 2019).

On the other hand, two-dimensional cancer cell lines are used in basic cancer research. Cell lines are also commercially available and available for canine bladder cancer. Although cell lines have been frequently used in studies such as the study of intracellular signal pathways due to their advantages such as ease of culture, low cost, and high growth speed, individual characteristics and cells such as organoids have been used. There was a problem that it could not reflect the diversity and polarity of the cells.

It has also been suggested that cancer stem cells contribute to the development of cancer, postoperative recurrence and treatment resistance. As a method for identifying cancer stem cell markers, the ability to form tumors by transplanting cells isolated by flow cytometry analysis or sorting method into immunodeficient mice and the ability to form spheres in an in vitro experimental system are used as indicators. It has been. However, specific cancer stem cell markers for each cancer type are still unclear, which poses a barrier to therapeutic and diagnostic applications.

Sato et al., Nature, 459 (7244): 262-5, 2009 Wetering et al., Cell, 161 (4): 933-45, 2015 Boj et al., Cell, 160 (1-2): 32-38, 2015 Elbadawy and Usui et al., Cancer Sci. 110 (9): 2806-2821, 2019

Conventional three-dimensional organoids can reflect original characteristics such as patient-derived cancer tissue, but have a problem of slow cell growth rate. On the other hand, cell lines that have acquired infinite proliferative capacity by immortalization have a high growth rate, but cannot maintain the diversity of cells that make up patient-derived cancer tissues, etc., so the obtained results are returned to clinical practice. It is difficult to do. In addition, the strains that can be established as a cell line are limited, and there is also a problem that it takes an enormous amount of time and labor to establish a cell line from cells contained in a specific biological sample.

Therefore, in view of the above-mentioned circumstances, the present invention reflects the original characteristics of patient-derived cancer tissues such as three-dimensional organoids, and at the same time, cells having a characteristic of high growth rate such as cell lines. It is an object of the present invention to provide a culture body and a method for producing the same.

As a result of diligent studies by the present inventors in order to achieve the above-mentioned object, a unique idea such as the two-dimensionalization of cells having a migration ability generated when culturing a three-dimensional organoid has been reached, and the three-dimensional organoid has. We have found that it is possible to prepare a cell culture that maintains its characteristics and exhibits a growth rate faster than that of a three-dimensional organoid, and has completed the present invention.

The present invention includes the following.
[1] A step of culturing a three-dimensional organoid prepared from a biological sample in a medium for animal cells, and leaving the cells having a migration ability derived from the three-dimensional organoid that adhere to a culture vessel, and components other than the cells. A method for producing a cell culture, which comprises forming a cell culture on the bottom surface of the culture container, which comprises a step of removing the cells from the culture container.
[2] The method for producing a cell culture according to [1], wherein the animal cell medium contains an EGF and a TGFβ inhibitor.
[3] The method for producing a cell culture according to [1], wherein the animal cell medium does not contain one or both of a Wnt agonist and a BMP inhibitor.
[4] The method for producing a cell culture according to the above [1], further comprising a step of subculturing the cells adhered to the culture vessel.
[5] The method for producing a cell culture according to the above [1], wherein the above-mentioned component is a component containing a non-adhesive cell contained in a three-dimensional organoid and a medium for a three-dimensional organoid.
[6] The method for producing a cell culture according to the above [1], wherein the biological sample is a sample containing cancer cells derived from animals.
[7] A cell culture produced by the production method according to any one of the above [1] to [6].
[8] A bioassay method for analyzing a gene expression pattern in a cell culture produced by the production method according to any one of the above [1] to [6].
[9] The bioassay method according to [8] above, which analyzes the gene expression pattern of a cancer-related gene.

The cell culture according to the present invention has an excellent feature that the cell proliferation rate is enhanced as compared with the three-dimensional organoid while maintaining the characteristics and properties of the three-dimensional organoid. Therefore, by using the cell culture body according to the present invention, it is possible to perform the same various bioassays as when using the three-dimensional organoid, and it is possible to proliferate more efficiently than culturing the three-dimensional organoid. ..

In addition, the method for producing a cell culture according to the present invention is a tertiary cell culture having excellent characteristics such as an increase in cell proliferation rate as compared with a three-dimensional organoid while maintaining the characteristics and properties of the three-dimensional organoid. It can be manufactured from the original organoid.

It is a schematic diagram which shows the process of preparation and analysis of the cell culture body which concerns on this invention. It is a phase contrast microscope image which shows the 2.5D bladder cancer organoid prepared in this Example. It is a phase-contrast microscope image showing a 2.5D bladder cancer organoid prepared from three-dimensional bladder cancer organoids of different strains. It is a phase-contrast microscope image when a 2.5D bladder cancer organoid was prepared using culture solutions having different compositions. It is a fluorescence micrograph showing the expression of the urothelial cell marker in the prepared 2.5D bladder cancer organoid. It is a characteristic diagram which shows cell proliferation rate of 2.5D bladder cancer organoid and three-dimensional organoid. It is a characteristic diagram which shows the expression of various cancer stem cell markers in 2.5D bladder cancer organoid. It is a photograph showing the tumorigenicity of 2.5D bladder cancer organoid in vivo. It is a photograph which shows the histopathological image of the excised 2.5D bladder cancer organoid-derived tumor. FIG. 3 is a fluorescence micrograph showing urothelial cell marker expression in an excised 2.5D bladder cancer organoid-derived tumor. It is a phase-contrast microscope image showing 2.5D bladder cancer organoid after treatment with various anticancer agents. It is a characteristic diagram which shows the survival rate of 2.5D bladder cancer organoid after treatment with various anticancer agents. It is a phase-contrast microscope image showing a 2.5D organoid prepared using a human colorectal cancer three-dimensional organoid.

Hereinafter, the present invention will be described in detail.
The cell culture according to the present invention is prepared from a three-dimensional organoid prepared from a biological sample, and has a characteristic that the growth rate is faster than that of the three-dimensional organoid while maintaining the characteristics and properties of the three-dimensional organoid. It is a group. Generally, a cell line (immortalized, infinite proliferation ability) prepared from a biological sample spreads on the bottom surface of a culture vessel and is two-dimensionally cultured. That is, the culture of the cell line is a two-dimensional culture having a spread in the plane direction. The cell culture according to the present invention has the characteristics and properties of a three-dimensional organoid as described above, but is a culture having a spread in the plane direction like a two-dimensional culture. Therefore, the cell culture according to the present invention can also be referred to as a 2.5-dimensional organoid (2.5D organoid).

Here, the biological sample means a sample containing pluripotent stem cells (PSC), adult stem cells (ASC), or cancer stem cells, which are necessary for producing a three-dimensional organoid. Examples of biological samples include cancer tissues, blood and urine samples collected from individuals suffering from cancer, biopsy samples obtained from various tissues such as liver, brain, and intestine. These biological samples are not particularly limited and can be obtained from mammals. Mammals include, but are not limited to, rabbits, mice, rats, cows, sheep, cats, dogs, horses, monkeys and humans.

For the three-dimensional organoid, a medium for producing organoids containing a Wnt agonist that activates Wnt signaling, a BMP inhibitor that inhibits BMP signaling, and epidermal growth factor (EGF) is used to support a scaffold such as an extracellular matrix. It is produced by culturing stem cells embedded in the body. The organoid preparation medium can maintain organ-specific stem cells and cancer stem cells by the above-mentioned components.

Examples of Wnt agonists include R-spondins such as Wnt, Wnt-3a, Noggin, GSK inhibitors, R-spondin 1, R-spondin 2, R-spondin 3 and R-spondin 4.

BMP inhibitors include Noggin, Chordin, chordin-like proteins containing chordin domains, hollistatin, holistatin-related proteins including hollistatin domains, DAN, DAN cysteine-DAN-like proteins including knot domains. , Sclerostin / SOST, decorin and α-2 macroglobulin and the like.

The culture conditions for producing the three-dimensional organoid are not particularly limited, and for example, Sato T et al., Gastroenterology. 2011 Nov; 141 (5): 1762-72 and the like can be referred to. For the preparation of bladder cancer organoids using urine samples of cancer-affected dogs, refer to Reference 1: Elbadawy and Usui et al., Cancer Sci. 110 (9): 2806-2821, 2019. can.

Examples of the scaffolding support used when producing a three-dimensional organoid include a polymer capable of absorbing and retaining water such as collagen and agarose, and a sponge-like membrane such as porous polystyrene. More specifically, as the scaffolding support, a collagen-containing matrigel (manufactured by Corning Life Science Co., Ltd.) can be used.

When preparing the cell culture according to the present invention, first, the three-dimensional organoids prepared as described above are cultured in a medium for animal cells (hereinafter referred to as a medium for 2.5D organoids). Here, the medium for 2.5D organoid is not particularly limited as long as it has a composition capable of culturing cells having migration ability derived from a three-dimensional organoid, and for example, a medium for animal cells containing an EGF and a TGFβ inhibitor may be used. can. The 2.5D organoid medium has a composition different from that of the three-dimensional organoid production medium. That is, the medium for producing three-dimensional organoids has a composition for maintaining tissue-specific stem cells and cancer stem cells embedded in the extracellular matrix, whereas the medium for 2.5D organoid has a composition for maintaining the stem cells. Is different. Specifically, the medium for 2.5D organoids is different in composition from the medium for producing three-dimensional organoids in that it does not contain one or both of Wnt agonists and BMP inhibitors.

EGF is a 6045 Da protein consisting of 53 amino acid residues and three intramolecular disulfide bonds that bind as a ligand to the epidermal growth factor receptor (EGFR) present on the cell surface. The concentration of EGF contained in the medium for 2.5D organoid is not particularly limited, but can be, for example, 2 ng / mL to 500 ng / mL, preferably 5 ng / mL to 500 ng / mL, and 10 ng / mL. It is more preferably ~ 400 ng / mL, more preferably 20 ng / mL to 300 ng / mL, more preferably 30 ng / mL to 200 ng / mL, and more preferably 40 ng / mL to 100 ng / mL. More preferred. More specifically, the concentration of EGF contained in the 2.5D organoid medium can be 50 ng / mL.

Examples of TGFβ (transforming growth factor β) inhibitors include A83-01 (3- (6-methylpyridin-2-yl) -1-phenylthiocarbamoyl-4-quinoline-4-ylpyrazol) and ALK5 Inhibitor I. (3- (Pyridine-2-yl) -4- (4-quinonyl) -1H-pyrazol), LDN193189 (4- (6- (4- (piperazin-1-yl) phenyl) pyrazolo [1,5-a ] Pyrimidine-3-yl) quinoline), SB431542 (4- [4- (1,3-benzodioxol-5-yl) -5-pyridin-2-yl-1H-imidazol-2-yl] benzamide) , SB-505124 (2- (5-benzo [1,3] dioxol-5-yl-2-tert-butyl-3H-imidazol-4-yl) -6-methylpyridin hydrochloride hydrate), SD-208 ((2- (5-Chloro-2-fluorophenyl) pteridine-4-yl) pyridine-4-yl-amine), SB-525334 (6- [2- (1,1-dimethylethyl) -5-( 6-Methyl-2-pyridinyl) -1H-imidazole-4-yl] quinoline), LY-364947 (4- [3- (2-pyridinyl) -1H-pyrazol-4-yl] -quinoline), LY2157299 (4) -[2- (6-Methyl-pyridin-2-yl) -5,6-dihydro-4H-pyrrolo [1,2-b] pyrazole-3-yl] -quinoline-6-carboxylic acid amide), TGF- β RI Kinase Inhibitor II 616452 (2- (3- (6-methylpyridin-2-yl) -1H-pyrazole-4-yl) -1,5-naphthylidine), TGF-β RI Kinase Inhibitor III 616453 (2-yl) (5-Benzo [1,3] dioxol-4-yl-2-tert-butyl-1H-imidazol-4-yl) -6-methylpyrididine, HCl), TGF-β RI Kinase Inhibitor IX 616463 (4-( (4-((2,6-Dimethylpyridin-3-yl) oxy) Pyridine-2-yl) amino) benzenesulfonamide), TGF-β RI Kinase Inhibitor VII 616458 (1-2-((6,7-yl)) Dimethoxy-4-quinolyl) oxy)-(4,5-dimethylphenyl) -1-etanone), naphthylidine (6- (2-tert-butyl-5- (6-methyl-pyridine-2-yl) -1H-imidazol-4-yl) -quinoxalin), AP12009 (TGF-β2 antisense compound "Trabedersen"), Belagenpumatucel -L (TGF-β2 antisense gene-modified allogeneic tumor cell vaccine), CAT-152 (Glaucoma-lerdelimumab (anti-TGF-β-2 monoclonal antibody)), CAT-192 (Metelimumab (human who neutralizes TGFβ1) IgG4 monoclonal antibody)), GC-1008 (anti-TGF-β monoclonal antibody) and the like.

2.5D Organoid Medium The concentration of the TGF-β inhibitor contained in the medium varies depending on the type and is not particularly limited. For example, when A83-01 is used as a TGF-β inhibitor, it can be 0.1 μM to 5 μM, preferably 0.2 μM to 3 μM, more preferably 0.3 μM to 1 μM. It is more preferably 0.4 μM to 0.8 μM. More specifically, the concentration of A83-01 contained in the 2.5D organoid medium can be 0.5 μM.

In addition to EGF and TGF-β inhibitors, the 2.5D organoid medium can contain various components contained in the medium used for normal animal cell culture. Examples of various components include amino acids such as N-acetyl-L-cysteine and L-glutamic acid and L-glutamine having an antioxidant effect, vitamins such as nicotinamide, and inorganic salts. A medium for 2.5D organoids can be prepared by adding EGF, TGF-β inhibitor and various components thereof to a basal medium generally used for animal cells. The medium for 2.5D organoid may contain fetal bovine serum (FBS) or fetal calf serum.

The cell culture medium used as the basal medium is preferably for animal cells or human cells. Examples of the basal medium include a synthetic medium having a pH of 7.0 to 7.6. More specifically, Dulbecco's Modified Eagle Medium (Nutrient Mixture F-12; DMEM / F12) and RPMI1640 medium (Roswell Park Memorial Institute 1640 medium) can be used. .. Further, as the basal medium, Advanced-DMEM / F12, Advanced RPMI medium and the like can also be used.

When culturing a three-dimensional organoid in a medium for 2.5D organoids, the culturing temperature can be 30 to 40 ° C, most preferably about 37 ° C. The culture time can be appropriately adjusted depending on the three-dimensional organoid used. A few days after the start of the culture, cell migration is induced, and the cells having the migration ability adhere to the bottom surface of the culture vessel or the like. More specifically, the migration ability is achieved by culturing the three-dimensional organoid using a medium for 2.5D organoids for about 3 to 14 days, preferably for about 5 to 10 days, and more preferably for about 6 to 8 days. It is possible to form a state in which the cells having the above are adhered to the bottom surface of the culture vessel or the like.

Then, the components excluding the cells adhered to the bottom surface of the culture vessel and the like are removed from the culture vessel. Here, the components to be removed include extracellular matrix, three-dimensional organoids formed in the cell matrix, cells having non-adherent migration ability, and the like. By removing the components other than the cells adhering to the bottom surface of the culture vessel from the culture vessel, a cell culture derived from a three-dimensional organoid can be formed on the bottom surface of the culture vessel or the like.

The obtained cell culture has a characteristic that it exhibits a faster growth rate than the three-dimensional organoid while maintaining the characteristics and properties of the three-dimensional organoid. The characteristics / properties of a three-dimensional organoid mean all or part of the properties of the tissue or cancer that is the source of the three-dimensional organoid, and for example, gene expression specific to the tissue or cancer that is the source of the organoid. It means that all or part of the pattern is preserved. Therefore, even in the cell culture prepared as described above, all or a part of the original tissue or cancer-specific gene expression pattern is preserved.

In addition, the cell culture obtained as described above can be subcultured in the same manner as a normal cell line. When the obtained cell culture is subcultured, it is preferable to use the above-mentioned 2.5D organoid medium.

The cell culture according to the present invention can be easily prepared from three-dimensional organoids by using the medium for 2.5D organoids as described above, and maintains the characteristics and properties of the three-dimensional organoids. On the other hand, when establishing a cell line from an organ or cancer tissue, it takes a huge amount of time and effort to clone a mutantly immortalized cell. As described above, the cell culture according to the present invention is characterized in that it can be produced very easily as compared with a normal cell line.

Further, since the cell culture according to the present invention has a higher growth rate than the three-dimensional organoid, the time required for subculture is short, and the cell culture can be obtained more efficiently than the three-dimensional organoid. Further, the medium used for preparing the cell culture according to the present invention and the medium used for subculture are compared with the medium used for preparing the three-dimensional organoid and the medium used for subculturing. It can be prepared very cheaply.

Furthermore, the cell culture according to the present invention can be subcultured simply by exchanging the medium in the same manner as a normal cell line. On the other hand, three-dimensional organoids require work such as melting a gel-like extracellular matrix to form a new extracellular matrix, and very complicated work is required for subculture. As described above, it can be said that the cell culture body according to the present invention is very easy to handle in subculture as compared with the three-dimensional organoid.

Therefore, by using the cell culture according to the present invention, it is possible to perform a bioassay similar to the bioassay using a three-dimensional organoid. That is, by using the cell culture according to the present invention for the bioassay, various bioassays based on the gene expression pattern in the original organ or cancer tissue can be performed more easily and easily as compared with the case where the three-dimensional organoid is used. It can be implemented at low cost.

Specifically, by using the cell culture according to the present invention, for example, the expression analysis of cancer markers, the analysis of tumorigenicity in vivo, and the analysis of responsiveness to anticancer agents can be performed in the same manner as for three-dimensional organoids. Can be carried out. For example, a susceptibility test to a known anticancer agent can be performed using the cell culture according to the present invention. Specifically, by preparing a cell culture using a three-dimensional organoid prepared from an animal to be tested such as a patient and performing a susceptibility test on the cell culture, the test result can be obtained for each animal to be tested. .. This makes it possible to individually consider the optimal treatment for cancer patients and animals suffering from cancer.

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

[Example 1]
[Preparation of three-dimensional bladder cancer organoid derived from urine sample]
The urine of a dog suffering from bladder cancer was collected using a catheter, the cells contained in the urine were washed, and then mixed with Matrigel for three-dimensional organoid culture. Three-dimensional bladder cancer organoids that proliferate three-dimensionally were observed about 3 to 14 days after the start of culture (Reference 1: Elbadawy & Usui et al., Cancer Science. 110 (9): 2806-2821, 2019. ). Similar to Reference 1, the formed three-dimensional bladder cancer organoid is analyzed for epithelial structure by H & E staining, observation of urinary tract epithelial cell marker expression by immunohistochemical staining, and reactivity to various anticancer agents by Alamablue assay. And evaluated the tumor remodeling ability by subcutaneous transplantation into immunodeficient mice, and confirmed that the characteristics of bladder cancer tissue in vivo can be reproduced.

[Cell culture, preparation of 2.5D bladder cancer organoid]
Next, an attempt was made to prepare a 2.5D organoid (cell culture according to the present invention) using a three-dimensional bladder cancer organoid derived from each patient that reproduced the characteristics of bladder cancer. First, a culture solution (2.5D organoid culture solution) was prepared by modifying the culture solution used for three-dimensional bladder cancer organoid culture (Table 1).

Since this culture medium does not contain the expensive stem cell signal supplements Wnt 3a, Noggin and R-spondin used in the conventional 3D organoid culture method, it can be prepared at a lower cost than the 3D organoid culture medium. It was observed that when the three-dimensional bladder cancer organoid was cultured for 3 to 14 days using this culture medium, cell migration from the three-dimensional bladder cancer organoid was induced, and gradually adhered to the culture dish and proliferated. (The arrowheads in FIGS. 1 and 2). Adherent cells were isolated and cultured as 2.5D bladder cancer organoids, and passages were repeated (Fig. 2). As a subculture method, cells were washed with PBS solution, treated with 5 mM EDTA / PBS solution, and allowed to stand in a _{CO 2 incubator for 10 minutes.} Then, trypsin was treated for 5 minutes to collect the cells, the supernatant was aspirated, mixed with a new culture medium, and the cell solution was added to the dish. In FIG. 2, Passege 1 is a 2.5D organoid cell that has been passaged once, Passege 3 is a 2.5D organoid cell that has been passaged three times, and Passege 13 is a 2.5D organoid cell that has been passaged 13 times. Is.

[Efficiency of 2.5D bladder cancer organoid production]
It was shown that the production of 2.5D bladder cancer organoids can be isolated and cultured from 3D bladder cancer organoids of various strains (BC1 to BC3), as shown in FIG.

[Examination of usefulness of 2.5D organoid culture solution]
In order to examine the usefulness of the 2.5D organoid culture medium, a comparison was made with the culture medium of a normal cell line (Dulbecco-modified Eagle's medium (see Table 1)). When the 2.5D organoid culture medium shown in Table 1 was used, cell adhesion and proliferation were observed even after passage from the three-dimensional bladder cancer organoid, but when using Dalbecco's modified Eagle medium, the cells were subcultured. It was found that the cells became difficult to adhere to each other and culture became impossible due to repeated generations (Fig. 4). From this result, it is shown that the culture medium having the composition shown in Table 1 is essential for the subculture of 2.5D organoids.

[Expression of urothelial cell markers in 2.5D bladder cancer organoids]
In order to investigate the composition of cells contained in the 2.5D bladder cancer organoid prepared in this example, the expression of urothelial cell markers was confirmed. 2.5D bladder cancer organoids showed expression of urothelial cell markers in both shallow passages (early passages: 4-8 passages) and multiple passages (late passages: 10-20 passages). (Fig. 5). FIG. 5 shows the results of immunofluorescent staining for the urothelial cell markers CK7, CK20 and UPK3A (see Reference 1 for the experimental protocol).

[Evaluation of cell proliferation speed of 2.5D bladder cancer organoid]
The cell proliferation rates of 2.5D bladder cancer organoids and three-dimensional bladder cancer organoids were compared and examined using the Prestoblue assay. Specifically, 2.5D organoid cells in early passage and late passage and 3D organoid cells of the same strain were simultaneously seeded on a 96-well plate, and the number of viable cells on days 1, 3 and 5 of culture was subjected to a presto blue assay. Used to measure and compare. The result is shown in FIG. As shown in FIG. 6, the 2.5D bladder cancer organoids in the early passage and the late passage showed a significant increase in the growth rate as compared with the three-dimensional bladder cancer organoids.

[Examination of cancer stem cell marker expression in 2.5D bladder cancer organoids]
The expression levels of bladder cancer stem cell markers in 2.5D bladder cancer organoids were examined in comparison with three-dimensional bladder cancer organoids and cell lines. Specifically, RNA was extracted from 2.5D organoid cells of early passage and late passage, three-dimensional organoid cells of the same lineage, and two-dimensional cell lines using the Nucleo Spin kit (Takarabio), and the QuantiTect Reverse Transcription Kit (QuantiTect Reverse Transcription Kit) After conversion to cDNA using QIAGEN), PCR was performed using QuantiTect SYBR I Kit (QIAGEN) and StepOnePlus Real-Time PCR System (Applied Biosystems) to compare and examine the expression of stem cell markers CD44 and SOX2. .. In this experiment, a canine urothelial cancer cell line (manufactured by Cosmo Bio Co., Ltd.) was used as the cell line. In this experiment, two strains (BC1 and BC2) were used as a three-dimensional bladder cancer organoid and a 2.5D bladder cancer organoid.

The result is shown in FIG. As shown in FIG. 7, it can be seen that the SOX2 expression level in the 2.5D bladder cancer organoids of early passage and late passage is maintained to some extent as compared with the original three-dimensional bladder cancer organoid. In contrast, in normal cell lines, SOX2 expression was significantly reduced. In addition, the expression level of CD44 was as low as that of the three-dimensional bladder cancer organoid in the 2.5D bladder cancer organoid in both the early passage and the late passage, but was remarkably high in the normal cell line. These results suggest that 2.5D organoids show a distribution pattern of stem cell marker expression close to that of three-dimensional organoids, and that the SOX2 gene may be the most useful index for stem cell markers in bladder cancer cells.

[Evaluation of tumor remodeling ability of 2.5D bladder cancer organoids in vivo]
We investigated whether 2.5D bladder cancer organoids have the same tumor remodeling ability in mice as 3D bladder cancer organoids. Specifically, 2.5D organoid cells were transplanted under the skin of the back of immunodeficient mice, and the tumor formed 8 weeks later was excised and various analyzes were performed.
The result is shown in FIG. As shown in FIG. 8, it was observed that tumor formation was formed in all individuals after 8 weeks by ^{subcutaneous transplantation (1 × 10 6 cells, N = 4) into immunodeficient mice.}

[Histopathological image of 2.5D bladder cancer organoid-derived excised tumor]
The tissue was pathologically observed using the 2.5D bladder cancer organoid-derived tumor tissue excised in the above experiment. Specifically, the excised tissue was embedded in paraffin and then H & E stained.
The result is shown in FIG. As shown in FIG. 9, pathological features of typical urothelial cancer were observed in 2.5D bladder cancer organoid-derived tumor tissue.

[Expression of urothelial cell markers in 2.5D bladder cancer organoid-derived excised tumors]
The expression of urothelial cell markers (CK7, CK20 and UPK3A) in the 2.5D bladder cancer organoid-derived tumor tissue excised in the above experiment was confirmed in the same manner as above. The results are shown in FIG. As shown in FIG. 10, expression of the urothelial cell marker was also observed in the 2.5D bladder cancer organoid-derived tumor tissue.

[Induction of cell death by treatment with various anticancer drugs for 2.5D bladder cancer organoids]
After inoculating 1000 2.5D bladder cancer organoids into 96-well plates, they were treated with the anti-cancer agents Vinblastine, Mitoxantrone and Carboplatin for 72 hours each. After that, the change to cell morphology was observed and the viability was measured using the Prestoblue assay.

The results of cell morphology observation are shown in FIG. 11, and the results of the presto blue assay are shown in FIG. As shown in FIG. 11, treatment with known anticancer agents vinblastine, mitoxantrone and carboplatin suppressed the growth of 2.5D bladder cancer organoids in a concentration-dependent manner, and an image was observed in which cell death was induced at high concentrations. .. In addition, as shown in FIG. 12, the 2.5D bladder cancer organoid showed almost the same reactivity to the anticancer drug as compared with the original three-dimensional organoid. This result was observed regardless of the number of passages of the 2.5D bladder cancer organoid.

[Preparation of 2.5D organoids derived from human colorectal cancer 3D organoids]
A 2.5D colorectal cancer organoid was also prepared from a three-dimensional organoid derived from a human colorectal cancer patient prepared using a colorectal cancer surgical sample performed at the Department of Gastroenterology, Yamaguchi University School of Medicine. Colorectal cancer three-dimensional culture organoids were prepared using the air liquid interface culture method (Usui et al., Stem cells international, 7053872 2016). Specifically, the 3D colorectal cancer organoid was treated with a 2.5D organoid culture solution, and 7 days later, the migrating cells were isolated and passaged repeatedly. In this example as well, the 2.5D organoid culture solution having the composition shown in Table 1 was used to prepare a 2.5D colorectal cancer organoid under the same conditions as when the 2.5D bladder cancer organoid was prepared.

The results are shown in FIG. As shown in FIG. 13, it was clarified that a 2.5D colorectal cancer organoid can be produced from human colorectal cancer.

Claims (9)

A process of culturing a three-dimensional organoid prepared from a biological sample in a medium for animal cells, and
Among the cells having migration ability derived from the above-mentioned three-dimensional organoid, the step of leaving the cells adhered to the culture vessel and removing the components other than the cells from the culture vessel is included.
A method for producing a cell culture, which comprises forming a cell culture on the bottom surface of the culture container. The method for producing a cell culture according to claim 1, wherein the animal cell medium contains an EGF and a TGFβ inhibitor. The method for producing a cell culture according to claim 1, wherein the animal cell medium does not contain one or both of a Wnt agonist and a BMP inhibitor. The method for producing a cell culture according to claim 1, further comprising a step of subculturing the cells adhered to the culture vessel. The method for producing a cell culture according to claim 1, wherein the component is a component containing a non-adhesive cell contained in a three-dimensional organoid and a medium for a three-dimensional organoid. The method for producing a cell culture according to claim 1, wherein the biological sample is a sample containing cancer cells derived from animals. A cell culture produced by the production method according to any one of claims 1 to 6. A bioassay method for analyzing a gene expression pattern in a cell culture produced by the production method according to any one of claims 1 to 6. The bioassay method according to claim 8, wherein the gene expression pattern of a cancer-related gene is analyzed.

Images