WO2016047801A1 - Method of preparing spheroids of primary cancer cells, spheroids, screening method, and determination method - Google Patents

Method of preparing spheroids of primary cancer cells, spheroids, screening method, and determination method Download PDF

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WO2016047801A1
WO2016047801A1 PCT/JP2015/077297 JP2015077297W WO2016047801A1 WO 2016047801 A1 WO2016047801 A1 WO 2016047801A1 JP 2015077297 W JP2015077297 W JP 2015077297W WO 2016047801 A1 WO2016047801 A1 WO 2016047801A1
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cells
spheroid
spheroids
medium
drug
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PCT/JP2015/077297
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French (fr)
Japanese (ja)
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弘 五字
伊藤 学
哲也 中面
真菜美 下村
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Jsr株式会社
国立研究開発法人国立がん研究センター
Scivaxライフサイエンス株式会社
Jsrライフサイエンス株式会社
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    • GPHYSICS
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    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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    • G01N33/5082Supracellular entities, e.g. tissue, organisms
    • G01N33/5085Supracellular entities, e.g. tissue, organisms of invertebrates
    • GPHYSICS
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    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
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    • C12N2500/00Specific components of cell culture medium
    • C12N2500/05Inorganic components
    • C12N2500/10Metals; Metal chelators
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    • G01N2500/10Screening for compounds of potential therapeutic value involving cells

Definitions

  • the present invention relates to a method for producing spheroids of primary cancer cells, a spheroid, a screening method, and a determination method.
  • the key to obtaining useful research results is how to suppress the mixing and proliferation of cells with a high growth rate.
  • a method for suppressing mixed fibroblasts a method of changing amino acids in a medium, a method using a medium characterized by serum-free and low calcium concentration, a method using a difference in sensitivity to enzymes, and a difference in specific gravity
  • a method of using, a method of using feeder cells, and the like are known (for example, see Non-Patent Document 1).
  • the problem to be solved by the present invention is to provide a culture environment advantageous for culturing cancer cells while suppressing the proliferation ability of cells having a high growth rate in a tissue mixed with cells having a high growth rate. It is an object of the present invention to provide a method for producing spheroids, in which spheroids containing cells derived from cancer cells as main components are obtained.
  • a method for producing spheroids of primary cancer cells comprising a step of preparing a medium containing at least 1% by volume of serum in a total volume, and a cell culture substrate subjected to a treatment for suppressing adhesion to cells A step of preparing, a step of separating cells from a tissue piece containing cancer cells, a step of seeding cells on the cell culture substrate in the medium, and culturing the cells.
  • ⁇ 2> A spheroid obtained by the spheroid production method of ⁇ 1> above.
  • a screening method for a substance that acts directly or indirectly on a spheroid comprising the step of obtaining the spheroid of ⁇ 2> and a step of administering a test substance to the spheroid.
  • a method for determining the sensitivity of a drug to a spheroid comprising: obtaining the spheroid according to ⁇ 2>above; and administering a drug to the spheroid and observing a reaction caused by the drug.
  • ⁇ 5> The step of obtaining the spheroid of the above ⁇ 2>, the step of administering a drug to the spheroid and observing the reaction due to the drug, and analyzing the gene sequence of cells sorted from a tissue fragment containing cancer cells And a method of determining the sensitivity of the drug to spheroids, comprising: a step of comparing the reaction by the drug with the result of gene sequence analysis.
  • a kit for producing spheroids of primary cancer cells comprising a medium containing at least 1% by volume or more of serum in the total volume, and a cell culture substrate subjected to a treatment for suppressing adhesion to cells
  • a kit comprising:
  • cells derived from primary cancer cells can be obtained by providing a culture environment advantageous for culturing cancer cells while suppressing the proliferation ability of cells having a high growth rate in tissues mixed with cells having a high growth rate. It was possible to provide a method for producing a spheroid that can obtain a spheroid contained as a main component.
  • the method for producing spheroids containing primary cancer cells of the present invention is a method for producing spheroids of primary cancer cells, comprising a step of preparing a medium containing at least 1% by volume of serum in the total volume, and suppressing adhesion to cells Preparing a cell culture substrate subjected to the treatment, sorting the cells from a tissue piece containing cancer cells, seeding the cells on the cell culture substrate in the medium, and culturing the cells And a step of performing.
  • a step of preparing a medium containing at least 1% by volume of serum in the total volume Preparing a cell culture substrate subjected to the treatment, sorting the cells from a tissue piece containing cancer cells, seeding the cells on the cell culture substrate in the medium, and culturing the cells And a step of performing.
  • the description of a to b and the like representing a range such as a numerical range is synonymous with “a” to “b”, and the numerical values of “a” and “b” are included in the range.
  • the spheroid in the present invention means an aggregate of cells in which cells are aggregated and aggregated three-dimensionally.
  • the method for producing spheroids containing primary cancer cells of the present invention includes the step of preparing a medium containing at least 1% by volume of serum in the entire volume.
  • any cell culture basic medium, differentiation medium, primary culture medium or the like can be used. Examples include Dulbecco's modified Eagle medium (DMEM), Glasgow MEM (GMEM), RPMI 1640, Ham F12, serum-free medium (MCDB medium, etc.), but are not limited thereto. Furthermore, serum, various growth factors, and differentiation-inducing factors may be added to these media.
  • DMEM Dulbecco's modified Eagle medium
  • GMEM Glasgow MEM
  • RPMI 1640 Ham F12
  • MCDB medium serum-free medium
  • serum various growth factors, and differentiation-inducing factors may be added to these media.
  • the medium contains at least 1% by volume of serum in the entire volume. Normally, when culturing primary cancer cells, a low serum or serum-free medium is used to suppress the growth of stromal cells, but in the present invention, one containing serum is used. Serum is not particularly limited, and examples include fetal serum such as bovine and horse, newborn serum, fetal serum and the like. Of these, fetal bovine serum (FBS) is preferred.
  • the concentration of the serum is preferably 1 to 20% by volume, more preferably 3 to 15% by volume, more preferably 5 to 10% by volume, with 100% by volume as the total of all components in the medium. preferable.
  • the medium preferably contains insulin. Insulin is included to promote protein synthesis and promote cell growth.
  • the concentration of the insulin is preferably 1 ⁇ g / mL to 100 ⁇ g / mL in the medium, more preferably 5 ⁇ g / mL to 50 ⁇ g / mL, and further preferably 5 ⁇ g / mL to 20 ⁇ g / mL.
  • the medium preferably further contains one or more selected from hydrocortisone and epidermal growth factor (EGF), and more preferably includes both of them.
  • the concentration of each component in the medium is preferably 1 to 5000 ng / mL, more preferably 1 to 100 ng / mL, and still more preferably 10 to 20 ng / mL.
  • the medium preferably further contains at least one selected from transferrin, sodium selenate, sodium selenite, sodium pyruvate, and glutamine, and is selected from transferrin, sodium selenite, sodium pyruvate, and glutamine. It is preferable that 1 or more types are included. In particular, it is preferable to include any of transferrin, sodium selenite, sodium pyruvate, and glutamine.
  • the concentration of transferrin in the medium is preferably 1 to 100 ⁇ g / mL, more preferably 1 to 50 ⁇ g / mL, and even more preferably 5 to 20 ⁇ g / mL.
  • the concentration of sodium selenate and sodium selenite in the medium is preferably 1 to 100 nM, more preferably 10 to 100 nM, and even more preferably 30 to 80 nM.
  • the concentration of sodium pyruvate in the medium is preferably 0.01 to 100 mM, more preferably 0.1 to 10 mM, and even more preferably 1 to 5 mM.
  • the concentration of glutamine in the medium is preferably 0.01 to 100 mM, more preferably 0.1 to 10 mM, and even more preferably 1 to 5 mM.
  • the medium may further contain one or more selected from ethanolamine, triiodothyronine, BSA, and phosphorylethanolamine.
  • the concentration in the medium is preferably 1 to 100 ⁇ M, more preferably 10 to 100 ⁇ M, and even more preferably 30 to 50 ⁇ M.
  • triiodothyronine its concentration in the medium is preferably 1 to 1000 pM, more preferably 5 to 500 pM, and even more preferably 5 to 100 pM.
  • BSA the concentration in the medium is preferably 0.1 to 100 mg / mL, more preferably 1 to 10 mg / mL, and even more preferably 2 to 5 mg / mL.
  • phosphorylethanolamine its concentration in the medium is preferably 1 to 100 ⁇ M, more preferably 5 to 100 ⁇ M, and even more preferably 5 to 20 ⁇ M.
  • the method for producing spheroids containing primary cancer cells of the present invention includes a step of preparing a cell culture substrate that has been subjected to treatment for suppressing adhesion to cells.
  • the cell culture substrate in the present invention is subjected to a treatment for suppressing adhesion to cells.
  • Suppression of adhesion with cells is performed by any one of (1) a predetermined uneven structure in which the upper surface of the convex portion functions as a cell adhesion surface, (2) advanced hydrophilic treatment, and (3) advanced hydrophobic treatment.
  • Cell adhesion may be suppressed by two or more of the above (1) to (3), but those having at least the uneven structure of (3) are preferred.
  • the convex part is comprised in order to inhibit that a cell adhere
  • the concavo-convex structure surface where the upper surface of the convex portion functions as a cell adhesion surface is a pillar shape, dot shape, line shape (line and space), hole shape, multiple continuous or non-continuous polygons, depending on the nature of the cells to be cultured.
  • the shape a structure in which a plurality of polygons are regularly arranged is better.
  • a structure in which a plurality of polygons are continuous as the shape in the planar direction can be used.
  • the shape in the plane direction is more preferably a regular polygon such as a regular triangle, a square, or a regular hexagon, or a circular shape in that a target cell can be grown on an isotropically uniform structure.
  • the width between the polygonal pattern structures is 20 ⁇ m or less from the viewpoint that cells are grown three-dimensionally (spheroid culture) or differentiated, and cultured in a state closer to the living body. 10 ⁇ m or less, 5 ⁇ m or less, 3 ⁇ m or less, 1 ⁇ m or less, 700 nm or less, or 500 nm or less, the smaller the better. This is because the smaller the width between the polygons, the more the cells adhered to the concavo-convex structure surface can form spheroids while growing many pseudopods.
  • the depth of the polygonal structure is formed in various sizes such as 1 nm or more, 10 nm or more, 100 nm or more, 200 nm or more, 500 nm or more, 1 ⁇ m or more, 10 ⁇ m or more, 100 ⁇ m or more depending on the properties of the cells to be cultured. It may be.
  • the aspect ratio of the unevenness includes various things such as 0.2 or more, 0.5 or more, 1 or more, 2 or more.
  • the minimum inner diameter of the polygon is preferably 3 ⁇ m or less, and is preferably as small as 2 ⁇ m or less, 1 ⁇ m or less, 700 nm or less, 500 nm or less, or 250 nm or less for the same reason as described above.
  • the maximum inner diameter of the polygon is preferably within this range.
  • the inner diameter means the distance between two parallel lines circumscribing the polygon
  • the minimum inner diameter means the shortest distance among the two parallel lines circumscribing the polygon
  • the maximum inner diameter means the longest distance between two parallel lines circumscribing a polygon.
  • the distance between the opposite parallel sides is the minimum inner diameter
  • the distance between the opposite vertices is the maximum inner diameter
  • the length of the short side is the minimum inner diameter
  • the length of the diagonal line is the maximum inner diameter
  • the advanced hydrophilic treatment for suppressing cell adhesion may be any treatment as long as cells can be cultured, but is performed by coating a hydrophilic compound, for example, Poly-HEMA coat, Hydrogel coat, Poly-PEG coating and phospholipid coating are performed.
  • a hydrophilic compound for example, Poly-HEMA coat, Hydrogel coat, Poly-PEG coating and phospholipid coating are performed.
  • the advanced hydrophobic treatment for suppressing cell adhesion may be any treatment capable of culturing cells, but is performed by coating a hydrophobic compound, for example, by fluorine coating.
  • the shape of the cell culture substrate in the production method of the present invention may be any shape as long as cells can be cultured. Examples thereof include a film shape and a substrate shape (plate shape). , Dish, multi-well plate, flask, chamber slide and the like. Moreover, the uneven structure should just be formed in at least one part on a base material.
  • the material of the cell culture substrate may be any material as long as it is non-toxic to cells.
  • polystyrene polyethylene”, “polypropylene”, “polyimide”, “polylactic acid, Biodegradable polymers such as lactic acid-polyglycolic acid copolymer and polycaprolactone ”,“ cyclic olefin thermoplastic resins such as cyclic olefin copolymer (COC) and cyclic olefin polymer (COP) ”,“ acrylic resin ” , “Other resins such as photo-curing resin and thermosetting resin”, “metal such as aluminum oxide”, “glass”, “quartz glass”, “silicon” and the like can be used.
  • a substrate body made of silicon, glass or the like on which a coating layer such as “resin”, “photoresist”, “metal such as aluminum oxide” is formed.
  • the uneven structure of the cell culture substrate in the production method of the present invention is controlled by adjusting the polarity. May be used. Examples of the adjustment method include the following methods, but are not limited thereto.
  • the surface of the culture substrate can be provided with a functional group such as —O or —OH group to adjust the polarity.
  • a functional group such as —O or —OH group
  • the substance which raises polarity can also adjust by using the substance which raises polarity, or the substance which reduces polarity.
  • substances that increase the polarity include silicon dioxide (SiO 2 ), polylysine, and extracellular matrix components.
  • the extracellular matrix include various collagens, proteoglycans, fibronectin, laminin, and elastin. It is done.
  • fluorine, silicon, polyhemaethyl polyacrylate, agar, or the like can be used as the substance that lowers the polarity.
  • Polarity can be adjusted by using these substances as a base material and coating the surface of the concavo-convex structure.
  • the planar shape, the width between cells, the material of the culture substrate, the polarity, etc. may be appropriately adjusted according to the cell type to be seeded. Thereby, it becomes possible to acquire the spheroid according to the objective.
  • the cell culture substrate may be produced by any method.
  • a method for forming the concavo-convex structure for example, a nanoimprint technique, a solution casting method, etching, blasting, corona discharge, or the like can be used.
  • a method using a nanoimprint technique is preferable in that the shape and the like can be controlled more precisely.
  • JP 2010-22366 A for example, JP 2010-22366 A.
  • JP-A-2008-296481 can be referred to.
  • the method for producing spheroids containing primary cancer cells of the present invention includes a step of sorting cells from a tissue piece containing cancer cells.
  • the cancer cell in the present invention may be any cancer cell in animals such as mammals.
  • Examples include cancer, rhabdomyosarcoma, skin cancer, anal cancer, other various cancer cells, various stem cells, various progenitor cells, mesenchymal progen
  • the cell is not limited to a single cell but may be an aggregate of a plurality of cell types.
  • animals including mammals, but animals belonging to primates including monkeys and humans, animals belonging to rodents such as mice, squirrels and rats, animals belonging to rabbits, cats such as dogs and cats, etc.
  • rodents such as mice, squirrels and rats
  • animals belonging to rabbits cats such as dogs and cats, etc.
  • An animal belonging to the eye is exemplified.
  • tissue pieces or cell groups excised from a living body can be used, and these are treated with enzyme treatment, density gradient centrifugation treatment, filter treatment, magnetic beads, flow cytometer as necessary. Further, it may be separated and purified by some other treatment.
  • These cell groups may be aggregates of cells derived from the same tissue and different in differentiation stage.
  • the method for producing spheroids containing primary cancer cells of the present invention includes a step (culturing step) of seeding cells on the cell culture substrate in the medium and culturing the cells.
  • the culture in the present invention can be carried out according to the same culture procedure as that in the usual operation, and may be any of stationary culture, aeration culture, shaking culture, stirring culture and the like.
  • the number of cancer cells to be seeded is preferably 10 to 160 cells / mm 2 , preferably 20 to 80 cells / mm 2 as the number of cells per bottom area of a cell culture substrate such as a well. More preferably, it is more preferably 30 to 40 pieces / mm 2 .
  • the culture temperature is preferably 20 to 45 ° C.
  • the pH during culture is preferably 7 to 8
  • the culture time is preferably 1 to 30 days.
  • the collection may be performed according to a conventional method.
  • the spheroids obtained by the method of the present invention can be used for drug screening, food functionality evaluation, drug or food safety evaluation, regenerative medicine, and the like.
  • the cancer cells when cancer cells and cells other than cancer cells coexist, the cancer cells can be easily and inexpensively formed in a living tissue by efficiently forming spheroids without being driven out by cells having a high growth rate. Since it is possible to create a close environment, it can be used in a wide range of technologies related to medical and biotechnology such as regenerative medicine, drug discovery screening, cell engineering, and tissue engineering. Specific examples of applications of the spheroids include the following screening methods and determination methods.
  • a method for screening a substance that acts directly or indirectly on a spheroid comprising the steps of obtaining the spheroid and administering a test substance to the spheroid.
  • a method for determining the sensitivity of a drug to a spheroid comprising: obtaining the spheroid; and administering a drug to the spheroid and observing a reaction due to the drug.
  • Such a determination method may further include a step of analyzing a gene sequence of a cell collected from a tissue piece containing cancer cells, and a step of collating a reaction by a drug with a result of gene sequence analysis.
  • the substance which acts directly or indirectly on the spheroid in the screening method is not particularly limited, and examples thereof include various compounds, antibodies, nucleic acids and the like including anticancer agents.
  • the drug used in the determination method may be an anticancer agent, for example, actinomycin D, melphalan, busulfan, carboplatin, cisplatin, cyclophosphamide, dacarbazine, oxaliplatin, procarbazine, temozolomide, ifosfamide, liposomal doxorubicin, Doxorubicin, daunorubicin, epirubicin, idarubicin, mitomycin C, bleomycin, mitoxantrone, cladribine, fluorouracil, mercaptopurine, pemetrexado, methotrexate, cytarabine, nelarabine, capecitabine, fludarabine, gemcitabine, pentostatin bricritine vincristine , Docetaxel, etoposide, vinorelbine, nogitecan, park Taxel, Tretinoin, Bevacizumab,
  • the kit of the present invention is a kit for producing spheroids of primary cancer cells, and is a cell culture that has been subjected to a treatment that suppresses adhesion between cells and a medium containing at least 1% by volume of serum in the total volume. And a substrate.
  • the medium and cell culture substrate include those used in the above spheroid production method.
  • sample acquisition and pretreatment A portion of lung cancer tissue removed from a patient who had given consent at the hospital was immediately transferred to a tube to which 10% FBS-containing DMEM medium was added and stored on ice.
  • the DMEM medium containing 10% FBS was removed, the tissue washing solution was added and removed three times to wash the tissue piece, and then the wet weight of the tissue was measured.
  • the tissue piece was placed in a 10 cm petri dish on ice, tissue washing solution (0.5 to 1 mL) was added and minced to 1 mm square with scissors, and then collected in a 50 mL tube to obtain a tissue suspension. Collagenase and dispase were added to the tissue suspension, followed by enzyme treatment at 37 ° C.
  • the amount of the reaction solution for enzyme treatment was 5 mL up to a tissue wet weight of 300 mg, and 10 mL for 301-600 mg.
  • the final concentration of each enzyme was collagenase (1 mg / mL) and dispase (1000 PU / mL). After the enzyme reaction, a part was collected, and after confirming the dispersibility of the cells / tissues, the number of cells was counted.
  • the reaction was weakened by adding a tissue washing solution twice the amount of the reaction solution, and tissue residues such as fibers were removed through a 100 ⁇ m mesh cell strainer.
  • the tube and the cell strainer were washed with an appropriate amount of tissue washing solution, and the cells were collected and centrifuged at 300 ⁇ g for 5 minutes. After removing the supernatant, the tissue washing solution (10 mL) was added to the pellet for resuspension and centrifuged at 300 ⁇ g for 5 min. Thereafter, the cells were resuspended with 1 to 3 mL of tissue washing solution, and the number of cells was counted.
  • Examples 1 to 7, Comparative Example 1 and Comparative Example 2 Cell seeding and culture Three-dimensional culture plate NCP-LS-96 (96 well, shape in the planar direction of the concavo-convex structure: square pattern) manufactured by SCIVAX Life Sciences, with various culture media (150 ⁇ L / well) having the composition shown in Table 1 After adding and centrifuging at 700 ⁇ g for 5 min at room temperature, the mixture was allowed to stand at 37 ° C. for 10 min and prewetting was performed.
  • a necessary amount of the counted cells is collected in a 1.5 mL tube, centrifuged at 300 ⁇ g for 5 minutes to remove the supernatant, and then cells having a concentration of 2 ⁇ 10 5 cells / mL in various culture media having the composition shown in Table 1.
  • a suspension was prepared. 100 ⁇ L thereof was seeded on a plate, and culture was started at 37 ° C. and 5% CO 2 . The number of seeded cells was 2 ⁇ 10 4 cells / 250 ⁇ L / well, the seeding date was day 0, and half of the medium was exchanged on days 1, 3 and 5.
  • CK19 antibody solution diluted 100-fold with TBST containing 3% BSA was added, and the primary antibody was reacted for 1 hr.
  • a negative control without antibody was also prepared.
  • 500 ⁇ L of TBST was added and centrifuged at 300 ⁇ g for 5 minutes, 500 ⁇ L of TBST was added again to the pellet from which the supernatant was removed, and the cells were washed at 300 ⁇ g for 5 minutes to wash the cells.
  • 50 ⁇ L of Alexa Fluor 488 goat anti-mouse IgG antibody solution diluted 1000 times with TBST containing 3% BSA was added, and the secondary antibody was reacted for 1 hr.
  • Example 8 Anticancer drug susceptibility test A cell suspension was prepared with the medium of Example 5 (hereinafter simply referred to as culture medium), and a three-dimensional culture plate NCP-LS-384 (384 well, concavo-convex structure plane made by SCIVAX Life Sciences) (Shape of direction: square pattern) was seeded under the conditions of 37 ° C., 5% CO 2 , 3000 cells / 80 ⁇ L / well (day 0). On day 1, 40 ⁇ L of the medium was removed from the wells seeded with cells, and 40 ⁇ L of new culture medium was added.
  • culture medium hereinafter simply referred to as culture medium
  • NCP-LS-384 384 well, concavo-convex structure plane made by SCIVAX Life Sciences
  • Example of results-The right figure in Fig. 1 is a sensitive EGFR exon19-deficient specimen and is known to be highly sensitive to gefetinib (anticancer drugs are effective) ⁇
  • the left figure of FIG. 1 is WT and is known to have low sensitivity to gefetinib (anticancer drugs do not work)
  • the sample on the right side of FIG. 1 has no EGF receptor gene mutation that is sensitive to gefetinib, indicating that the sensitivity of gefetinib by this method is weak.
  • the sample in the left figure of FIG. 1 is a sample in which the nucleotide sequence of exon 19 of the EGFR-coding gene, which has been found to be sensitive to gefetinib, is shown to be less sensitive to gefetinib by this method. It was.

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Abstract

Provided is a method of preparing spheroids by which spheroids including, as a main component, cells derived from primary cancer cells are obtained by inhibiting the proliferating ability of cells having a rapid proliferation rate and by providing a culturing environment favorable for culturing cancer cells, in tissue in which the cells having a rapid proliferating rate have been incorporated. The method for preparing spheroids of primary cancer cells is characterized by including the steps for: preparing a culture medium including at least 1 vol% of serum with respect to the total volume thereof; preparing a cell culture substrate on which a treatment for inhibiting adhesiveness to cells is performed; isolating cells from a tissue fragment including cancer cells; and seeding the cells on the cell culture substrate in the culture medium and culturing the cells.

Description

初代癌細胞のスフェロイド作製方法、スフェロイド、スクリーニング方法、及び、判定方法Primary cancer cell spheroid preparation method, spheroid, screening method, and determination method
 本発明は、初代癌細胞のスフェロイド作製方法、スフェロイド、スクリーニング方法、及び、判定方法に関する。 The present invention relates to a method for producing spheroids of primary cancer cells, a spheroid, a screening method, and a determination method.
 今日、創薬や再生医学に関わる研究開発の発展に伴い、生体組織の機能をより正しく理解するための研究の動きが急速に高まっている。生体組織機能の理解には、その組織を構成する細胞の性質を理解することが重要である。細胞レベルでの研究の手段としては、株化されている細胞を用いる系と、組織から目的とする細胞を直接取り出して培養する初代培養系がある。初代培養系では、個体から取り出した細胞を用いていることから、細胞そのものの形質および機能や細胞集団の構成が、生体内の状態を比較的良く反映していると考えられ、生体内で起きている現象の解明に非常に大きな意義を持つ。 Today, with the development of research and development related to drug discovery and regenerative medicine, the movement of research to understand the function of living tissues more rapidly is rapidly increasing. To understand the function of living tissue, it is important to understand the nature of the cells that make up the tissue. As a means of research at the cell level, there are a system using established cells and a primary culture system in which target cells are directly taken out from a tissue and cultured. In primary culture systems, cells taken from individuals are used, so the characteristics and functions of the cells themselves and the composition of the cell population are considered to reflect the state of the living body relatively well. It has great significance for the elucidation of the phenomenon.
 初代培養系においては、増殖速度の速い細胞の混入・増殖をいかに抑えるかが、有用な研究結果を得る鍵となっている。例えば、混入した線維芽細胞の抑制方法として、培地中のアミノ酸を変更する方法、無血清・低カルシウム濃度を特徴とする培地を用いる方法、酵素に対する感受性の差を利用する方法、比重の違いを利用する方法、フィーダー細胞を用いる方法等が知られている(例えば、非特許文献1参照)。 In primary culture systems, the key to obtaining useful research results is how to suppress the mixing and proliferation of cells with a high growth rate. For example, as a method for suppressing mixed fibroblasts, a method of changing amino acids in a medium, a method using a medium characterized by serum-free and low calcium concentration, a method using a difference in sensitivity to enzymes, and a difference in specific gravity A method of using, a method of using feeder cells, and the like are known (for example, see Non-Patent Document 1).
特開2010-22366号公報JP 2010-22366 A
 本発明が解決しようとする課題は、増殖速度の早い細胞が混入した組織において、増殖速度の早い細胞の増殖能を抑制しつつ、癌細胞の培養に有利な培養環境を提供することにより、初代癌細胞由来の細胞を主要成分として含むスフェロイドが得られる、スフェロイド作製方法を提供することである。 The problem to be solved by the present invention is to provide a culture environment advantageous for culturing cancer cells while suppressing the proliferation ability of cells having a high growth rate in a tissue mixed with cells having a high growth rate. It is an object of the present invention to provide a method for producing spheroids, in which spheroids containing cells derived from cancer cells as main components are obtained.
 上記課題は、下記の手段により解決された。
 <1>初代癌細胞のスフェロイド作製方法であって、全体積中少なくとも1体積%以上の血清を含む培地を用意する工程と、細胞との接着性を抑制する処理を施した細胞培養基材を用意する工程と、癌細胞を含む組織片から細胞を分取する工程と、前記培地中、前記細胞培養基材上に細胞を播種し、前記細胞を培養する工程と、を含むことを特徴とする初代癌細胞のスフェロイド作製方法。
 <2>上記<1>のスフェロイド作製方法により得たことを特徴とする、スフェロイド。
 <3>上記<2>のスフェロイドを得る工程と、前記スフェロイドに対して被験物質を投与する工程と、を含むことを特徴とするスフェロイドに直接的又は間接的に作用を及ぼす物質のスクリーニング方法。
 <4>上記<2>のスフェロイドを得る工程と、前記スフェロイドに対して薬剤を投与し、薬剤による反応を観察する工程と、を含むことを特徴とするスフェロイドに対する前記薬剤の感受性の判定方法。
 <5>上記<2>のスフェロイドを得る工程と、前記スフェロイドに対して薬剤を投与し、薬剤による反応を観察する工程と、がん細胞を含む組織片から分取した細胞の遺伝子配列を分析する工程と、薬剤による反応と遺伝子配列分析結果を照合する工程と、を含むことを特徴とするスフェロイドに対する前記薬剤の感受性の判定方法。
 <6>初代癌細胞のスフェロイドを作製するためのキットであって、全体積中少なくとも1体積%以上の血清を含む培地と、細胞との接着性を抑制する処理を施した細胞培養基材とを備える、キット。
The above problems have been solved by the following means.
<1> A method for producing spheroids of primary cancer cells, comprising a step of preparing a medium containing at least 1% by volume of serum in a total volume, and a cell culture substrate subjected to a treatment for suppressing adhesion to cells A step of preparing, a step of separating cells from a tissue piece containing cancer cells, a step of seeding cells on the cell culture substrate in the medium, and culturing the cells. To produce spheroids of primary cancer cells.
<2> A spheroid obtained by the spheroid production method of <1> above.
<3> A screening method for a substance that acts directly or indirectly on a spheroid, comprising the step of obtaining the spheroid of <2> and a step of administering a test substance to the spheroid.
<4> A method for determining the sensitivity of a drug to a spheroid, comprising: obtaining the spheroid according to <2>above; and administering a drug to the spheroid and observing a reaction caused by the drug.
<5> The step of obtaining the spheroid of the above <2>, the step of administering a drug to the spheroid and observing the reaction due to the drug, and analyzing the gene sequence of cells sorted from a tissue fragment containing cancer cells And a method of determining the sensitivity of the drug to spheroids, comprising: a step of comparing the reaction by the drug with the result of gene sequence analysis.
<6> A kit for producing spheroids of primary cancer cells, comprising a medium containing at least 1% by volume or more of serum in the total volume, and a cell culture substrate subjected to a treatment for suppressing adhesion to cells A kit comprising:
 本発明により、増殖速度の早い細胞が混入した組織において、増殖速度の早い細胞の増殖能を抑制しつつ、癌細胞の培養に有利な培養環境を提供することにより、初代癌細胞由来の細胞を主要成分として含むスフェロイドが得られる、スフェロイド作製方法を提供することができた。 According to the present invention, cells derived from primary cancer cells can be obtained by providing a culture environment advantageous for culturing cancer cells while suppressing the proliferation ability of cells having a high growth rate in tissues mixed with cells having a high growth rate. It was possible to provide a method for producing a spheroid that can obtain a spheroid contained as a main component.
ゲフェチニブに対する感受性を示す図である。It is a figure which shows the sensitivity with respect to gefetinib. 初代肺がん細胞の抗がん剤感受性試験の結果を示す図である。It is a figure which shows the result of the anticancer agent sensitivity test of a primary lung cancer cell.
<初代癌細胞を含むスフェロイド作製方法>
 本発明の初代癌細胞を含むスフェロイド作製方法は、初代癌細胞のスフェロイド作製方法であって、全体積中少なくとも1体積%以上の血清を含む培地を用意する工程と、細胞との接着性を抑制する処理を施した細胞培養基材を用意する工程と、癌細胞を含む組織片から細胞を分取する工程と、前記培地中、前記細胞培養基材上に細胞を播種し、前記細胞を培養する工程と、を含むことを特徴とする。
 以下、各工程について説明する。なお、本発明において数値範囲等の範囲を表すa~b等の記載は、a以上、b以下と同義であり、a及びbの数値をその範囲内に含む。また、本明細書では、本発明におけるスフェロイドとは、三次元的に細胞同士が集合・凝集化した細胞の集合体を意味する。
<Method for producing spheroids containing primary cancer cells>
The method for producing spheroids containing primary cancer cells of the present invention is a method for producing spheroids of primary cancer cells, comprising a step of preparing a medium containing at least 1% by volume of serum in the total volume, and suppressing adhesion to cells Preparing a cell culture substrate subjected to the treatment, sorting the cells from a tissue piece containing cancer cells, seeding the cells on the cell culture substrate in the medium, and culturing the cells And a step of performing.
Hereinafter, each step will be described. In the present invention, the description of a to b and the like representing a range such as a numerical range is synonymous with “a” to “b”, and the numerical values of “a” and “b” are included in the range. In the present specification, the spheroid in the present invention means an aggregate of cells in which cells are aggregated and aggregated three-dimensionally.
(培地準備工程)
 本発明の初代癌細胞を含むスフェロイド作製方法は、全体積中少なくとも1体積%以上の血清を含む培地を用意する工程を含む。
(Medium preparation process)
The method for producing spheroids containing primary cancer cells of the present invention includes the step of preparing a medium containing at least 1% by volume of serum in the entire volume.
 培地としては、任意の細胞培養基本培地や分化培地、初代培養専用培地等を用いることができる。例えば、ダルベッコ改変イーグル培地(DMEM)、グラスゴーMEM(GMEM)、RPMI1640、ハムF12、無血清培地(MCDB培地等)等が挙げられるが、これらに限定されるものではない。さらに、これらの培地に血清や各種増殖因子、分化誘導因子を添加してもよい。 As the medium, any cell culture basic medium, differentiation medium, primary culture medium or the like can be used. Examples include Dulbecco's modified Eagle medium (DMEM), Glasgow MEM (GMEM), RPMI 1640, Ham F12, serum-free medium (MCDB medium, etc.), but are not limited thereto. Furthermore, serum, various growth factors, and differentiation-inducing factors may be added to these media.
 また、培地は、全体積中少なくとも1体積%以上の血清を含む。通常であれば、初代癌細胞を培養する場合には、間質系細胞の増殖を抑制するため低血清又は無血清培地が用いられるが、本発明においては血清を含むものを用いる。血清は特に限定されないが、ウシ、ウマ等の胎児血清や新生児血清、仔血清等が挙げられる。中でも、ウシ胎児血清(FBS)が好ましい。
 前記血清の濃度は、培地中の全成分合計を100体積%として1~20体積%であることが好ましく、3~15体積%であることがより好ましく、5~10体積%であることがさらに好ましい。
The medium contains at least 1% by volume of serum in the entire volume. Normally, when culturing primary cancer cells, a low serum or serum-free medium is used to suppress the growth of stromal cells, but in the present invention, one containing serum is used. Serum is not particularly limited, and examples include fetal serum such as bovine and horse, newborn serum, fetal serum and the like. Of these, fetal bovine serum (FBS) is preferred.
The concentration of the serum is preferably 1 to 20% by volume, more preferably 3 to 15% by volume, more preferably 5 to 10% by volume, with 100% by volume as the total of all components in the medium. preferable.
 培地は、インスリンを含むものが好ましい。インスリンを含むことで、タンパク質合成を促進し細胞の増殖を促す。前記インスリンの濃度は、培地中、1μg/mL~100μg/mLであることが好ましく、5μg/mL~50μg/mLであることがより好ましく、5μg/mL~20μg/mLであることがさらに好ましい。 The medium preferably contains insulin. Insulin is included to promote protein synthesis and promote cell growth. The concentration of the insulin is preferably 1 μg / mL to 100 μg / mL in the medium, more preferably 5 μg / mL to 50 μg / mL, and further preferably 5 μg / mL to 20 μg / mL.
 前記培地は、さらにヒドロコルチゾン及び上皮成長因子(EGF)から選ばれる1種以上を含むことが好ましく、これらの両方を含むことがより好ましい。各成分の培地中の濃度は、それぞれ、1~5000ng/mLであることが好ましく、1~100ng/mLであることがより好ましく、10~20ng/mLであることがさらに好ましい。 The medium preferably further contains one or more selected from hydrocortisone and epidermal growth factor (EGF), and more preferably includes both of them. The concentration of each component in the medium is preferably 1 to 5000 ng / mL, more preferably 1 to 100 ng / mL, and still more preferably 10 to 20 ng / mL.
 前記培地は、さらにトランスフェリン、セレン酸ナトリウム、亜セレン酸ナトリウム、ピルビン酸ナトリウム、及びグルタミンから選ばれる1種以上を含むことが好ましく、トランスフェリン、亜セレン酸ナトリウム、ピルビン酸ナトリウム、及びグルタミンから選ばれる1種以上を含むことが好ましい。特に、トランスフェリン、亜セレン酸ナトリウム、ピルビン酸ナトリウム、及びグルタミンのいずれをも含むのが好ましい。
 トランスフェリンの培地中の濃度は、1~100μg/mLであることが好ましく、1~50μg/mLであることがより好ましく、5~20μg/mLであることがさらに好ましい。
 セレン酸ナトリウム、亜セレン酸ナトリウムの培地中の濃度は、1~100nMであることが好ましく、10~100nMであることがより好ましく、30~80nMであることがさらに好ましい。
 ピルビン酸ナトリウムの培地中の濃度は、0.01~100mMであることが好ましく、0.1~10mMであることがより好ましく、1~5mMであることがさらに好ましい。
 グルタミンの培地中の濃度は、0.01~100mMであることが好ましく、0.1~10mMであることがより好ましく、1~5mMであることがさらに好ましい。
The medium preferably further contains at least one selected from transferrin, sodium selenate, sodium selenite, sodium pyruvate, and glutamine, and is selected from transferrin, sodium selenite, sodium pyruvate, and glutamine. It is preferable that 1 or more types are included. In particular, it is preferable to include any of transferrin, sodium selenite, sodium pyruvate, and glutamine.
The concentration of transferrin in the medium is preferably 1 to 100 μg / mL, more preferably 1 to 50 μg / mL, and even more preferably 5 to 20 μg / mL.
The concentration of sodium selenate and sodium selenite in the medium is preferably 1 to 100 nM, more preferably 10 to 100 nM, and even more preferably 30 to 80 nM.
The concentration of sodium pyruvate in the medium is preferably 0.01 to 100 mM, more preferably 0.1 to 10 mM, and even more preferably 1 to 5 mM.
The concentration of glutamine in the medium is preferably 0.01 to 100 mM, more preferably 0.1 to 10 mM, and even more preferably 1 to 5 mM.
 前記培地は、さらにエタノールアミン、トリヨードサイロニン(triiudothyronine)、BSA、及びフォスフォリルエタノールアミン(Phosphorylethanolamine)から選ばれる1種以上を含んでいてもよい。
 エタノールアミンを用いる場合、その培地中の濃度は、1~100μMであることが好ましく、10~100μMであることがより好ましく、30~50μMであることがさらに好ましい。
 トリヨードサイロニンを用いる場合、その培地中の濃度は、1~1000pMであることが好ましく、5~500pMであることがより好ましく、5~100pMであることがさらに好ましい。
 BSAを用いる場合、その培地中の濃度は、0.1~100mg/mLであることが好ましく、1~10mg/mLであることがより好ましく、2~5mg/mLであることがさらに好ましい。
 フォスフォリルエタノールアミンを用いる場合、その培地中の濃度は、1~100μMであることが好ましく、5~100μMであることがより好ましく、5~20μMであることがさらに好ましい。
The medium may further contain one or more selected from ethanolamine, triiodothyronine, BSA, and phosphorylethanolamine.
When ethanolamine is used, the concentration in the medium is preferably 1 to 100 μM, more preferably 10 to 100 μM, and even more preferably 30 to 50 μM.
When triiodothyronine is used, its concentration in the medium is preferably 1 to 1000 pM, more preferably 5 to 500 pM, and even more preferably 5 to 100 pM.
When BSA is used, the concentration in the medium is preferably 0.1 to 100 mg / mL, more preferably 1 to 10 mg / mL, and even more preferably 2 to 5 mg / mL.
When phosphorylethanolamine is used, its concentration in the medium is preferably 1 to 100 μM, more preferably 5 to 100 μM, and even more preferably 5 to 20 μM.
(細胞培養基材準備工程)
 本発明の初代癌細胞を含むスフェロイド作製方法は、細胞との接着性を抑制する処理を施した細胞培養基材を用意する工程を含む。
 本発明における細胞培養基材は、細胞との接着性を抑制する処理を施したものである。細胞との接着の抑制は、(1)凸部上面が細胞接着面として機能する所定の凹凸構造、(2)高度な親水処理、(3)高度な疎水処理のいずれかによって行われる。また、上記(1)~(3)のうち2種以上によって細胞接着が抑制されていてもよいが、上記(3)の凹凸構造を少なくとも有するものが好ましい。なお、凸部は、細胞が平面(凸部上面以外の平面)上に接着するのを阻害するために構成されている。
 凸部上面が細胞接着面として機能する凹凸構造面は、培養する細胞の性質に応じて、ピラー状、ドット状、ライン状(ラインアンドスペース)、ホール状、複数の連続したまたは連続しない多角形のパターン構造等、種々の形状(なお、この形状は細胞培養基材を上方から見たときの形状(平面方向の形状)をいう)とすることができるが、好ましくは、所定の平面方向の形状として多角形が規則的に複数配列した構造の方が良い。例えば、平面方向の形状として多角形が複数連続した構造とすることができる。この時、等方的に均一な構造上で目的細胞を成長させることができるという点で、平面方向の形状は、正三角形、正方形、正六角形等の正多角形や、円形のものがより好ましい。また、ピラー状やホール状の凹凸構造と多角形状の凹凸構造とを組み合わせることも可能である。
(Cell culture substrate preparation process)
The method for producing spheroids containing primary cancer cells of the present invention includes a step of preparing a cell culture substrate that has been subjected to treatment for suppressing adhesion to cells.
The cell culture substrate in the present invention is subjected to a treatment for suppressing adhesion to cells. Suppression of adhesion with cells is performed by any one of (1) a predetermined uneven structure in which the upper surface of the convex portion functions as a cell adhesion surface, (2) advanced hydrophilic treatment, and (3) advanced hydrophobic treatment. Cell adhesion may be suppressed by two or more of the above (1) to (3), but those having at least the uneven structure of (3) are preferred. In addition, the convex part is comprised in order to inhibit that a cell adhere | attaches on a plane (planes other than the upper surface of a convex part).
The concavo-convex structure surface where the upper surface of the convex portion functions as a cell adhesion surface is a pillar shape, dot shape, line shape (line and space), hole shape, multiple continuous or non-continuous polygons, depending on the nature of the cells to be cultured. It is possible to adopt various shapes such as the pattern structure (note that this shape refers to the shape when the cell culture substrate is viewed from above (the shape in the planar direction)), but preferably in a predetermined planar direction As the shape, a structure in which a plurality of polygons are regularly arranged is better. For example, a structure in which a plurality of polygons are continuous as the shape in the planar direction can be used. At this time, the shape in the plane direction is more preferably a regular polygon such as a regular triangle, a square, or a regular hexagon, or a circular shape in that a target cell can be grown on an isotropically uniform structure. . It is also possible to combine a pillar-shaped or hole-shaped uneven structure with a polygonal uneven structure.
 上記多角形のパターン構造間の幅は、細胞を単層状ではなく三次元的に成長させたり(スフェロイド培養)、分化させたりし、より生体内に近い状態で培養するという観点からは、20μm以下、10μm以下、5μm以下、3μm以下、1μm以下、700nm以下、500nm以下というように、小さくなるほど好ましい。この理由としては、多角形間の幅が小さくなるほど、凹凸構造面に接着した細胞は、多くの仮足を成長させながらスフェロイドを形成させることができると考えられるためである。 The width between the polygonal pattern structures is 20 μm or less from the viewpoint that cells are grown three-dimensionally (spheroid culture) or differentiated, and cultured in a state closer to the living body. 10 μm or less, 5 μm or less, 3 μm or less, 1 μm or less, 700 nm or less, or 500 nm or less, the smaller the better. This is because the smaller the width between the polygons, the more the cells adhered to the concavo-convex structure surface can form spheroids while growing many pseudopods.
 また、多角形構造の深さは、培養する細胞の性質に応じて、1nm以上、10nm以上、100nm以上、200nm以上、500nm以上、1μm以上、10μm以上、100μm以上等種々の大きさに形成されていてよい。 Further, the depth of the polygonal structure is formed in various sizes such as 1 nm or more, 10 nm or more, 100 nm or more, 200 nm or more, 500 nm or more, 1 μm or more, 10 μm or more, 100 μm or more depending on the properties of the cells to be cultured. It may be.
 また、この凹凸のアスペクト比としては、0.2以上、0.5以上、1以上、2以上等種々のものがある。 Further, the aspect ratio of the unevenness includes various things such as 0.2 or more, 0.5 or more, 1 or more, 2 or more.
 また、多角形の最小内径は、3μm以下であることが好ましく、2μm以下、1μm以下、700nm以下、500nm以下、250nm以下というように、小さくなるほど、上述同様の理由により好ましい。また、多角形の最大内径もこの範囲であるのが好ましい。
 ここで、内径とは、多角形に外接する2本の平行線間の距離を意味し、最小内径とは、多角形に外接する二本の平行線間の距離のうち最も短いものを言い、最大内径とは、多角形に外接する二本の平行線間の距離のうち最も長いものを言う。例えば、多角形が正六角形の場合には、対向する平行な辺と辺との間の距離が最小内径となり、対向する頂点間の距離が最大内径となる。また、多角形が長方形の場合には、短辺の長さが最小内径となり、対角線の長さが最大内径となる。
Further, the minimum inner diameter of the polygon is preferably 3 μm or less, and is preferably as small as 2 μm or less, 1 μm or less, 700 nm or less, 500 nm or less, or 250 nm or less for the same reason as described above. The maximum inner diameter of the polygon is preferably within this range.
Here, the inner diameter means the distance between two parallel lines circumscribing the polygon, and the minimum inner diameter means the shortest distance among the two parallel lines circumscribing the polygon, The maximum inner diameter means the longest distance between two parallel lines circumscribing a polygon. For example, when the polygon is a regular hexagon, the distance between the opposite parallel sides is the minimum inner diameter, and the distance between the opposite vertices is the maximum inner diameter. Further, when the polygon is a rectangle, the length of the short side is the minimum inner diameter, and the length of the diagonal line is the maximum inner diameter.
 細胞接着の抑制を行う高度な親水処理とは、細胞を培養できるものであればどのような処理でも良いが、親水性の化合物をコーティングすることによって行い、例えば、Poly-HEMAコート、Hydrogelコート、Poly-PEGコート、リン脂質コートによって行われる。 The advanced hydrophilic treatment for suppressing cell adhesion may be any treatment as long as cells can be cultured, but is performed by coating a hydrophilic compound, for example, Poly-HEMA coat, Hydrogel coat, Poly-PEG coating and phospholipid coating are performed.
 細胞接着の抑制を行う高度な疎水処理とは、細胞を培養できるものであればどのような処理でも良いが、疎水性の化合物をコーティングすることによって行い、例えば、フッ素コートによって行われる。 The advanced hydrophobic treatment for suppressing cell adhesion may be any treatment capable of culturing cells, but is performed by coating a hydrophobic compound, for example, by fluorine coating.
 本発明の作製方法における細胞培養基材の形状は、細胞を培養できるものであればどのような形状でも良いが、例えば、フィルム状や基板状(プレート状)が挙げられ、基材は、シャーレ、ディッシュ、マルチウェルプレート、フラスコ、チェンバースライド等のいずれでもよい。また、凹凸構造は、基材上の少なくとも一部に形成されていればよい。 The shape of the cell culture substrate in the production method of the present invention may be any shape as long as cells can be cultured. Examples thereof include a film shape and a substrate shape (plate shape). , Dish, multi-well plate, flask, chamber slide and the like. Moreover, the uneven structure should just be formed in at least one part on a base material.
 また、細胞培養基材の材質は、細胞に対し無毒性のものであればどのようなものでも良く、例えば、「ポリスチレン」、「ポリエチレン」、「ポリプロピレン」、「ポリイミド」、「ポリ乳酸やポリ乳酸-ポリグリコール酸共重合体、ポリカプロラクトン等の生分解性ポリマー」、「環状オレフィン共重合体(COC)や環状オレフィン重合体(COP)等の環状オレフィン系熱可塑性樹脂」、「アクリル樹脂」、「光硬化性樹脂や熱硬化性樹脂等のその他の樹脂」、「酸化アルミニウム等の金属」、「ガラス」、「石英ガラス」、「シリコン」等を用いることができる。また、シリコンやガラス等からなる基板本体の表面に、「樹脂」、「フォトレジスト」、「酸化アルミニウム等の金属」等の被覆層が形成されたものを用いることもできる。 The material of the cell culture substrate may be any material as long as it is non-toxic to cells. For example, “polystyrene”, “polyethylene”, “polypropylene”, “polyimide”, “polylactic acid, Biodegradable polymers such as lactic acid-polyglycolic acid copolymer and polycaprolactone ”,“ cyclic olefin thermoplastic resins such as cyclic olefin copolymer (COC) and cyclic olefin polymer (COP) ”,“ acrylic resin ” , “Other resins such as photo-curing resin and thermosetting resin”, “metal such as aluminum oxide”, “glass”, “quartz glass”, “silicon” and the like can be used. Further, it is also possible to use a substrate body made of silicon, glass or the like on which a coating layer such as “resin”, “photoresist”, “metal such as aluminum oxide” is formed.
 細胞は、親水性表面に接着し易く、疎水性表面には接着し難いことが知られているため、本発明の作製方法における細胞培養基材の凹凸構造は、極性の調節により親水性の制御がなされているものであってもよい。調節方法としては、下記に示す方法が挙げられるが、これらに限定されるものではない。 Since it is known that cells adhere easily to a hydrophilic surface and difficult to adhere to a hydrophobic surface, the uneven structure of the cell culture substrate in the production method of the present invention is controlled by adjusting the polarity. May be used. Examples of the adjustment method include the following methods, but are not limited thereto.
 例えば、紫外線、電子線、ガンマ線、プラズマ等の照射による表面改質技術により、培養基材表面に例えば-Oや-OH基といった官能基を持たせ、極性を調節することができる。これにより、細胞が接着する凹凸構造面の極性を上げることができる。 For example, by using a surface modification technique by irradiation with ultraviolet rays, electron beams, gamma rays, plasma, etc., the surface of the culture substrate can be provided with a functional group such as —O or —OH group to adjust the polarity. Thereby, the polarity of the concavo-convex structure surface to which the cells adhere can be increased.
 また、極性を上げる物質または極性を下げる物質を用いることにより調節することもできる。極性を上げる物質としては、例えば、二酸化ケイ素(SiO2)、ポリリジンの他、細胞外マトリックス成分等を用いることができ、細胞外マトリックスとしては、各種コラーゲン、プロテオグリカン、フィブロネクチン、ラミニン、エラスチン等が挙げられる。極性を下げる物質としては、フッ素、シリコン、ポリヘマ(poly2-hydroxyethylmethacrylate)、アガー等を用いることができる。これらの物質を基材材料として用いること、また、凹凸構造面上に被覆させることにより、極性の調節が可能となる。 Moreover, it can also adjust by using the substance which raises polarity, or the substance which reduces polarity. Examples of substances that increase the polarity include silicon dioxide (SiO 2 ), polylysine, and extracellular matrix components. Examples of the extracellular matrix include various collagens, proteoglycans, fibronectin, laminin, and elastin. It is done. As the substance that lowers the polarity, fluorine, silicon, polyhemaethyl polyacrylate, agar, or the like can be used. Polarity can be adjusted by using these substances as a base material and coating the surface of the concavo-convex structure.
 なお、本発明における細胞培養基材の凹凸構造面上は、播種する細胞種に応じて、平面形状、セル間の幅、培養基材の材質、極性等を適宜調節して行うのが良い。これにより、目的に応じたスフェロイドを取得することが可能となる。 It should be noted that on the concavo-convex structure surface of the cell culture substrate in the present invention, the planar shape, the width between cells, the material of the culture substrate, the polarity, etc. may be appropriately adjusted according to the cell type to be seeded. Thereby, it becomes possible to acquire the spheroid according to the objective.
 細胞培養基材の製造方法は、いかなる方法でもよい。凹凸構造を形成し得る方法としては、例えば、ナノインプリント技術、溶液キャスト法、エッチング、ブラスト、コロナ放電等を用いることができる。この時、より精密に形状等を制御できる点で、ナノインプリント技術による方法が好ましい。ナノインプリント技術による製法については、例えば、特開2010-22366号公報を参照することができる。溶液キャスト法による製法については、例えば、特開2008-296481号公報を参照することができる。 The cell culture substrate may be produced by any method. As a method for forming the concavo-convex structure, for example, a nanoimprint technique, a solution casting method, etching, blasting, corona discharge, or the like can be used. At this time, a method using a nanoimprint technique is preferable in that the shape and the like can be controlled more precisely. For the production method using the nanoimprint technique, reference can be made to, for example, JP 2010-22366 A. For the production method by the solution casting method, for example, JP-A-2008-296481 can be referred to.
(癌細胞分取工程)
 本発明の初代癌細胞を含むスフェロイド作製方法は、癌細胞を含む組織片から細胞を分取する工程を含む。
(Cancer cell sorting process)
The method for producing spheroids containing primary cancer cells of the present invention includes a step of sorting cells from a tissue piece containing cancer cells.
 本発明における癌細胞は、哺乳類をはじめとする動物における、いかなるがん細胞であっても良いが、例えば、リンパ腫、骨髄腫、脳腫瘍、乳癌、子宮体癌、子宮頚癌、卵巣癌、食道癌、胃癌、虫垂癌、大腸癌、肝細胞癌、胆嚢癌、胆管癌、膵臓癌、副腎癌、消化管間質腫瘍、中皮腫、喉頭癌、口腔底癌、歯肉癌、舌癌、頬粘膜癌、唾液腺癌、副鼻腔癌、上顎洞癌、前頭洞癌、篩骨洞癌、蝶型骨洞癌、甲状腺癌、腎臓癌、肺癌、骨肉腫、前立腺癌、精巣腫瘍、腎細胞癌、膀胱癌、横紋筋肉腫、皮膚癌、肛門癌、その他各種癌細胞、各種幹細胞、各種前駆細胞、間葉系前駆細胞および、ES細胞、iPS細胞等が挙げられる。なお、細胞は単一の細胞に限らず、複数の細胞種の集合体であっても良い。哺乳類をはじめとする動物に特に限定はないが、サルやヒトを含む霊長目に属する動物、マウス、リス、ラットなどのげっ歯目に属する動物、ウサギ目に属する動物、イヌ、ネコなどのネコ目に属する動物が例示される。 The cancer cell in the present invention may be any cancer cell in animals such as mammals. For example, lymphoma, myeloma, brain tumor, breast cancer, endometrial cancer, cervical cancer, ovarian cancer, esophageal cancer , Stomach cancer, appendix cancer, colon cancer, hepatocellular carcinoma, gallbladder cancer, bile duct cancer, pancreatic cancer, adrenal cancer, gastrointestinal stromal tumor, mesothelioma, laryngeal cancer, oral floor cancer, gingival cancer, tongue cancer, buccal mucosa Cancer, salivary gland cancer, sinus cancer, maxillary sinus cancer, frontal sinus cancer, ethmoid sinus cancer, sphenoid sinus cancer, thyroid cancer, kidney cancer, lung cancer, osteosarcoma, prostate cancer, testicular cancer, renal cell carcinoma, bladder Examples include cancer, rhabdomyosarcoma, skin cancer, anal cancer, other various cancer cells, various stem cells, various progenitor cells, mesenchymal progenitor cells, ES cells, iPS cells, and the like. The cell is not limited to a single cell but may be an aggregate of a plurality of cell types. There are no particular restrictions on animals including mammals, but animals belonging to primates including monkeys and humans, animals belonging to rodents such as mice, squirrels and rats, animals belonging to rabbits, cats such as dogs and cats, etc. An animal belonging to the eye is exemplified.
 本発明の作製方法における細胞としては、例えば、生体から摘出した組織片、細胞群を用いることができ、これらを必要に応じて酵素処理、密度勾配遠心処理、フィルター処理、磁気ビーズ、フローサイトメーター、その他なんらかの処理により分離精製したものであってもよい。なお、これらの細胞群は、同じ組織に由来し、分化段階の異なる細胞の集合体であってもよい。 As the cells in the production method of the present invention, for example, tissue pieces or cell groups excised from a living body can be used, and these are treated with enzyme treatment, density gradient centrifugation treatment, filter treatment, magnetic beads, flow cytometer as necessary. Further, it may be separated and purified by some other treatment. These cell groups may be aggregates of cells derived from the same tissue and different in differentiation stage.
(培養工程)
 本発明の初代癌細胞を含むスフェロイド作製方法は、前記培地中、前記細胞培養基材上に細胞を播種し、前記細胞を培養する工程(培養工程)を含む。本発明における培養は、通常行われる操作と同様の培養手順により実施することができ、静置培養、通気培養、振盪培養、撹拌培養等のいずれでもよい。
 培養工程において、癌細胞の播種細胞数は、ウェル等の細胞培養基材の底面積あたりの細胞数として、10~160個/mm2とするのが好ましく、20~80個/mm2とするのがより好ましく、30~40個/mm2とするのがさらに好ましい。
 培養温度は20~45℃が好ましく、培養するときのpHは7~8が好ましく、培養時間は、1~30日間が好ましい。
(Culture process)
The method for producing spheroids containing primary cancer cells of the present invention includes a step (culturing step) of seeding cells on the cell culture substrate in the medium and culturing the cells. The culture in the present invention can be carried out according to the same culture procedure as that in the usual operation, and may be any of stationary culture, aeration culture, shaking culture, stirring culture and the like.
In the culturing step, the number of cancer cells to be seeded is preferably 10 to 160 cells / mm 2 , preferably 20 to 80 cells / mm 2 as the number of cells per bottom area of a cell culture substrate such as a well. More preferably, it is more preferably 30 to 40 pieces / mm 2 .
The culture temperature is preferably 20 to 45 ° C., the pH during culture is preferably 7 to 8, and the culture time is preferably 1 to 30 days.
 スフェロイドを回収する場合、その回収は、常法に従い行えばよい。 When collecting spheroids, the collection may be performed according to a conventional method.
<スフェロイド、スクリーニング方法、判定方法>
 このようにして本発明方法により得られたスフェロイドは、薬剤スクリーニング、食品機能性評価、薬品または食品の安全性評価、再生医療等に使用することができる。
 本発明は、癌細胞と癌細胞以外の細胞が混在する場合に、癌細胞が増殖速度の速い細胞に駆逐されることなく、効率よくスフェロイドを形成することにより、簡便かつ安価に生体内組織に近い環境を作出することを可能とするため、再生医療、創薬のスクリーニング、細胞工学、組織工学などの医療・バイオテクノロジーに関わる広範な技術に使用できる。上記スフェロイドの応用例としては、具体的には、以下のスクリーニング方法、判定方法等が挙げられる。
 前記スフェロイドを得る工程と、前記スフェロイドに対して被験物質を投与する工程と、を含むことを特徴とするスフェロイドに直接的又は間接的に作用を及ぼす物質のスクリーニング方法。
 前記スフェロイドを得る工程と、前記スフェロイドに対して薬剤を投与し、薬剤による反応を観察する工程と、を含むことを特徴とするスフェロイドに対する前記薬剤の感受性の判定方法。斯かる判定方法は、がん細胞を含む組織片から分取した細胞の遺伝子配列を分析する工程と、薬剤による反応と遺伝子配列分析結果を照合する工程とをさらに含んでいてもよい。
 前記スクリーニング方法、判定方法は、スフェロイドを得る工程以外については常法に従って行えばよい。
 なお、スクリーニング方法におけるスフェロイドに直接的又は間接的に作用を及ぼす物質は特に限定されないが、抗癌剤をはじめとして、各種化合物、抗体、核酸等が挙げられる。判定方法に使用する薬剤は、抗癌剤であればよく、例えば、アクチノマイシンD、メルファラン、ブスルファン、カルボプラチン、シスプラチン、シクロホスファミド、ダカルバジン、オキサリプラチン、プロカルバジン、テモゾロミド、イホスファミド、リポソーマルドキソルビシン、ドキソルビシン、ダウノルビシン、エピルビシン、イダルビシン、マイトマイシンC、ブレオマイシン、ミトキサントロン、クラドリビン、フルオロウラシル、メルカプトプリン、ペメトレキサド、メトトレキサート、シタラビン、ネララビン、カペシタビン、フルダラビン、ゲムシタビン、ペントスタチン、ビンクリスチン、エリブリン、パクリタキセル、ビンブラスチン、イリノテカン、ドセタキセル、エトポシド、ビノレルビン、ノギテカン、パクリタキセル、トレチノイン、ベバシズマブ、トラスツズマブ、パニツムマブ、セツキシマブ、イブリツモマブチウキセタン、リツキシマブ、ゲムツズマブオゾガマイシン、エベロリムス、エルロチニブ、ラパチニブ、ゲフィチニブ、イマチニブ、ダサチニブ、スニチニブ、ソラフェニブ、ボルテゾミブ、タミバロテン、ニムスチン、ラニムスチン、エノシタビン、カルモフール、シタラビンオクフォスファート、テガフール、テガフール・ウラシル、テガフール・ギメラシル・オテラシルカリウム、ドキシフルリジン、ヒドロキシカルハミド、ソブゾキサン、ビンデシン、アクラルビシン、アムルビシン、ジノスタチンスチマラマー、ピラルビシン、ぺプロマイシン、ネダプラチン等が挙げられる。
<Spheroid, screening method, determination method>
Thus, the spheroids obtained by the method of the present invention can be used for drug screening, food functionality evaluation, drug or food safety evaluation, regenerative medicine, and the like.
In the present invention, when cancer cells and cells other than cancer cells coexist, the cancer cells can be easily and inexpensively formed in a living tissue by efficiently forming spheroids without being driven out by cells having a high growth rate. Since it is possible to create a close environment, it can be used in a wide range of technologies related to medical and biotechnology such as regenerative medicine, drug discovery screening, cell engineering, and tissue engineering. Specific examples of applications of the spheroids include the following screening methods and determination methods.
A method for screening a substance that acts directly or indirectly on a spheroid, comprising the steps of obtaining the spheroid and administering a test substance to the spheroid.
A method for determining the sensitivity of a drug to a spheroid, comprising: obtaining the spheroid; and administering a drug to the spheroid and observing a reaction due to the drug. Such a determination method may further include a step of analyzing a gene sequence of a cell collected from a tissue piece containing cancer cells, and a step of collating a reaction by a drug with a result of gene sequence analysis.
What is necessary is just to perform the said screening method and the determination method according to a conventional method except the process of obtaining a spheroid.
In addition, the substance which acts directly or indirectly on the spheroid in the screening method is not particularly limited, and examples thereof include various compounds, antibodies, nucleic acids and the like including anticancer agents. The drug used in the determination method may be an anticancer agent, for example, actinomycin D, melphalan, busulfan, carboplatin, cisplatin, cyclophosphamide, dacarbazine, oxaliplatin, procarbazine, temozolomide, ifosfamide, liposomal doxorubicin, Doxorubicin, daunorubicin, epirubicin, idarubicin, mitomycin C, bleomycin, mitoxantrone, cladribine, fluorouracil, mercaptopurine, pemetrexado, methotrexate, cytarabine, nelarabine, capecitabine, fludarabine, gemcitabine, pentostatin bricritine vincristine , Docetaxel, etoposide, vinorelbine, nogitecan, park Taxel, Tretinoin, Bevacizumab, Trastuzumab, Panitumumab, Cetuximab, Ibritumomab Tiuxetane, Rituximab, Gemtuzumab Ozogamicin, Everolimus, Erlotinib, Lapatinib, Gefitinib, Imatinib, Dasatinib Ranimustine, enositabine, carmofur, cytarabine okfosfert, tegafur, tegafur uracil, tegafur gimeracil oteracil potassium, doxyfluridine, hydroxycarbamide, sobuzoxan, vindesine, aclarubicin, amrubicin, dinostatin stimamer, pirarubicin And nedaplatin.
<キット>
 本発明のキットは、初代癌細胞のスフェロイドを作製するためのキットであって、全体積中少なくとも1体積%以上の血清を含む培地と、細胞との接着性を抑制する処理を施した細胞培養基材とを備えるものである。培地、細胞培養基材としては、上記スフェロイド作製方法で使用するものが挙げられる。
<Kit>
The kit of the present invention is a kit for producing spheroids of primary cancer cells, and is a cell culture that has been subjected to a treatment that suppresses adhesion between cells and a medium containing at least 1% by volume of serum in the total volume. And a substrate. Examples of the medium and cell culture substrate include those used in the above spheroid production method.
 以下、実施例を挙げて本発明を詳細に説明するが、本発明はこれら実施例に限定されるものではない。 Hereinafter, the present invention will be described in detail with reference to examples, but the present invention is not limited to these examples.
<検体入手及び前処理>
 病院において同意を得られた患者より摘出した肺がん組織の一部について、摘出後速やかに10%FBS含有DMEM培地を添加したチューブに移し、氷上にて保管した。分散処理に際しては、10%FBS含有DMEM培地を除去した後、組織洗浄液を添加・除去を3回繰り返して組織片を洗浄した後、組織の湿重量を測定した。
 組織片を氷上の10cmシャーレにとり、組織洗浄液(0.5~1mL)を添加してハサミで1mm角になるまでミンスした後、これを50mLのチューブに回収し、組織懸濁液とした。
 前記組織懸濁液にコラゲナーゼ及びディスパーゼを添加した後、ウォーターバスで震盪させながら37℃ 30minで酵素処理を行った。酵素処理の反応液量は組織の湿重量300mgまでは5mL、301~600mgは10mLとした。各酵素終濃度はコラゲナーゼ(1mg/mL)及びディスパーゼ(1000PU/mL)とした。酵素反応後、一部を回収し、細胞・組織の分散性を確認した後、細胞数をカウントした。反応液の2倍量の組織洗浄液を加えて反応を弱め、100μmメッシュのセルストレーナーを通し、線維等の組織残渣を除去した。適量の組織洗浄液でチューブ及びセルストレーナーを洗いこみ、細胞を回収して300×g、5min遠心した。上清を除去した後、ペレットに組織洗浄液(10mL)を加えて再懸濁し、300×g、5minで遠心した。その後、1~3mLの組織洗浄液で再懸濁し、細胞数のカウントを行った。
<Sample acquisition and pretreatment>
A portion of lung cancer tissue removed from a patient who had given consent at the hospital was immediately transferred to a tube to which 10% FBS-containing DMEM medium was added and stored on ice. In the dispersion treatment, the DMEM medium containing 10% FBS was removed, the tissue washing solution was added and removed three times to wash the tissue piece, and then the wet weight of the tissue was measured.
The tissue piece was placed in a 10 cm petri dish on ice, tissue washing solution (0.5 to 1 mL) was added and minced to 1 mm square with scissors, and then collected in a 50 mL tube to obtain a tissue suspension.
Collagenase and dispase were added to the tissue suspension, followed by enzyme treatment at 37 ° C. for 30 minutes while shaking in a water bath. The amount of the reaction solution for enzyme treatment was 5 mL up to a tissue wet weight of 300 mg, and 10 mL for 301-600 mg. The final concentration of each enzyme was collagenase (1 mg / mL) and dispase (1000 PU / mL). After the enzyme reaction, a part was collected, and after confirming the dispersibility of the cells / tissues, the number of cells was counted. The reaction was weakened by adding a tissue washing solution twice the amount of the reaction solution, and tissue residues such as fibers were removed through a 100 μm mesh cell strainer. The tube and the cell strainer were washed with an appropriate amount of tissue washing solution, and the cells were collected and centrifuged at 300 × g for 5 minutes. After removing the supernatant, the tissue washing solution (10 mL) was added to the pellet for resuspension and centrifuged at 300 × g for 5 min. Thereafter, the cells were resuspended with 1 to 3 mL of tissue washing solution, and the number of cells was counted.
(実施例1~7、比較例1及び比較例2)
 細胞播種及び培養
 SCIVAXライフサイエンス社製の3次元培養プレートNCP-LS-96(96well、凹凸構造の平面方向の形状:スクエアパターン)に、表1に示す組成の各種培養培地(150μL/well)を添加し、室温で、700×g、5min遠心した後、37℃、10min静置してプレウェッティングした。カウントした細胞を1.5mLチューブに必要量分取し、300×g、5min遠心して上清を除去した後、表1に示す組成の各種培養培地で2×105cells/mLの濃度の細胞懸濁液を調製した。その100μLをプレートに播種し、37℃、5%CO2条件で培養を開始した。播種細胞数は、2×104cells/250μL/wellであり、播種日をday 0とし、半量の培地交換をday1、3及び5に実施した。
(Examples 1 to 7, Comparative Example 1 and Comparative Example 2)
Cell seeding and culture Three-dimensional culture plate NCP-LS-96 (96 well, shape in the planar direction of the concavo-convex structure: square pattern) manufactured by SCIVAX Life Sciences, with various culture media (150 μL / well) having the composition shown in Table 1 After adding and centrifuging at 700 × g for 5 min at room temperature, the mixture was allowed to stand at 37 ° C. for 10 min and prewetting was performed. A necessary amount of the counted cells is collected in a 1.5 mL tube, centrifuged at 300 × g for 5 minutes to remove the supernatant, and then cells having a concentration of 2 × 10 5 cells / mL in various culture media having the composition shown in Table 1. A suspension was prepared. 100 μL thereof was seeded on a plate, and culture was started at 37 ° C. and 5% CO 2 . The number of seeded cells was 2 × 10 4 cells / 250 μL / well, the seeding date was day 0, and half of the medium was exchanged on days 1, 3 and 5.
(培養後の生細胞数増加率(%)測定)
 Day 1における生細胞数に対するDay 7での生細胞数増加率(%)を求めた。生細胞増加率の算出はATPアッセイ(CellTiter-Gloを使用)により行った。結果を表1に示す。
(Measurement of the rate of increase in the number of living cells after culture (%)
The increase rate (%) of the number of viable cells in Day 7 with respect to the number of viable cells in Day 1 was determined. The rate of increase in viable cells was calculated by ATP assay (using CellTiter-Glo). The results are shown in Table 1.
(癌細胞含有率(%,CK19陽性率))
 Day 1、7において、1.5mLチューブに回収した細胞を300×g、5min遠心して上清を除去したペレットに、Accumax(100μL)を加え、37℃で30min静置し、スフェロイドを分散させた。この細胞分散液に組織洗浄液を1mL加え、300×g、5min遠心した。上清を除去した後、ペレットに10%中性緩衝ホルマリンを100μL加えて15min静置し、細胞を固定した。固定後、組織洗浄液を1mL加え、300×g、5min遠心した。上清を除去後、組織洗浄液1mLで再懸濁し、染色まで4℃で冷蔵保存した。300×g、5min遠心後、上清を除去し、ペレットに0.1%Triton X-100含有TBS(50μL)加え、15min静置した。その後、3%BSA含有TBSTを450μL加え、300×g、5min遠心した。上清を除去して50μLの3%BSA含有TBSTで懸濁し、1hr静置し、ブロッキングを行った。ブロッキング後、3%BSA含有TBSTで100倍希釈したCK19抗体液を50μL添加し、1次抗体を1hr反応させた。この際、抗体なしのネガティブコントロールも調製した。反応後、TBST500μLを添加して300×g、5min遠心し、上清を除去したペレットに再度TBST500μL添加して300×g、5min遠心し、細胞を洗浄した。次に3%BSA含有TBSTで1000倍希釈したAlexa Fluor 488 goat anti-mouse IgG抗体液を50μL添加し、2次抗体を1hr反応させた。反応後、3%BSA含有TBST500μL添加して300×g、5min遠心し、上清を除去したペレットに再度TBST500μL添加して300×g、5min遠心し、細胞を洗浄した。最後に3%BSA含有TBSTで1μg/mLに希釈したDAPIを100μL添加した。
 各サンプルを蛍光イメージング用のプレートに移し、CK19および核染色の蛍光観察を行い、DAPI陽性細胞とCK19陽性細胞のカウントを行い、DAPI陽性細胞数に対するCK19陽性細胞数のカウントを行った。このCK19陽性細胞率を癌細胞含有率とした。結果を表1に示す。なお、表1中のFBSの濃度は、体積%濃度である。
(Cancer cell content (%, CK19 positive rate))
In Day 1 and 7, Accumax (100 μL) was added to the pellet from which the supernatant was removed by centrifugation at 300 × g for 5 min. . 1 mL of the tissue washing solution was added to the cell dispersion, and centrifuged at 300 × g for 5 minutes. After removing the supernatant, 100 μL of 10% neutral buffered formalin was added to the pellet and allowed to stand for 15 min to fix the cells. After fixation, 1 mL of tissue washing solution was added and centrifuged at 300 × g for 5 minutes. After removing the supernatant, it was resuspended with 1 mL of tissue washing solution and stored refrigerated at 4 ° C. until staining. After centrifugation at 300 × g for 5 min, the supernatant was removed, 0.1% Triton X-100-containing TBS (50 μL) was added to the pellet, and the mixture was allowed to stand for 15 min. Thereafter, 450 μL of 3% BSA-containing TBST was added and centrifuged at 300 × g for 5 minutes. The supernatant was removed, suspended in 50 μL of 3% BSA-containing TBST, and allowed to stand for 1 hr for blocking. After blocking, 50 μL of CK19 antibody solution diluted 100-fold with TBST containing 3% BSA was added, and the primary antibody was reacted for 1 hr. At this time, a negative control without antibody was also prepared. After the reaction, 500 μL of TBST was added and centrifuged at 300 × g for 5 minutes, 500 μL of TBST was added again to the pellet from which the supernatant was removed, and the cells were washed at 300 × g for 5 minutes to wash the cells. Next, 50 μL of Alexa Fluor 488 goat anti-mouse IgG antibody solution diluted 1000 times with TBST containing 3% BSA was added, and the secondary antibody was reacted for 1 hr. After the reaction, 500 μL of 3% BSA-containing TBST was added and centrifuged at 300 × g for 5 minutes, and 500 μL of TBST was added again to the pellet from which the supernatant had been removed, followed by centrifugation at 300 × g for 5 minutes to wash the cells. Finally, 100 μL of DAPI diluted to 1 μg / mL with TBST containing 3% BSA was added.
Each sample was transferred to a plate for fluorescence imaging, fluorescence observation of CK19 and nuclear staining was performed, DAPI positive cells and CK19 positive cells were counted, and the number of CK19 positive cells was counted relative to the number of DAPI positive cells. This CK19 positive cell rate was defined as the cancer cell content rate. The results are shown in Table 1. In addition, the density | concentration of FBS in Table 1 is a volume% density | concentration.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
(実施例8)
 抗がん剤感受性試験
 実施例5の培地(以下、単に培養培地という)で細胞懸濁液を調製し、SCIVAXライフサイエンス社製の3次元培養プレートNCP-LS-384(384well、凹凸構造の平面方向の形状:スクエアパターン)に、37℃、5%CO2、3000cells/80μL/wellの条件で播種した(day 0)。
 day 1に、細胞を播種したwellより培地を40μL除去し、新しい培養培地を40μL添加した。
 day 3に、培地を40μL除去し、新しい培養培地で目的の濃度(2及び10μmol/L)に希釈したゲフィチニブ溶液を40μL添加した。ゲフィチニブの最終濃度は1及び5μmol/Lとなる。また、コントロールとして、培養培地のみ添加した実験も行った。
 day 7に各wellから細胞を1.5mLチューブに回収し、ATP濃度を測定した。コントロールサンプルのATP濃度を100%とし、ゲフェチニブ添加群のATP濃度比を細胞生存率とした。
(Example 8)
Anticancer drug susceptibility test A cell suspension was prepared with the medium of Example 5 (hereinafter simply referred to as culture medium), and a three-dimensional culture plate NCP-LS-384 (384 well, concavo-convex structure plane made by SCIVAX Life Sciences) (Shape of direction: square pattern) was seeded under the conditions of 37 ° C., 5% CO 2 , 3000 cells / 80 μL / well (day 0).
On day 1, 40 μL of the medium was removed from the wells seeded with cells, and 40 μL of new culture medium was added.
To day 3, 40 μL of the medium was removed, and 40 μL of gefitinib solution diluted with the new culture medium to the target concentration (2 and 10 μmol / L) was added. The final concentration of gefitinib will be 1 and 5 μmol / L. As a control, an experiment in which only the culture medium was added was also conducted.
On day 7, cells from each well were collected in a 1.5 mL tube, and the ATP concentration was measured. The ATP concentration of the control sample was 100%, and the ATP concentration ratio of the gefetinib added group was the cell viability.
(EGFR遺伝子変異検体の同定)
 HE染色標本により腫瘍細胞が確認されたホルマリン固定パラフィン包埋組織ブロックより、10μmの厚さの連続切片にて未染標本スライド作製し、室温保存した。検体は脱パラフィン操作を行った後、コバスDNAプレバレーションキットを用いて検体内のDNA抽出を行った。EGFR遺伝子変異の検出はコバスEGFR変位検出キットを用いて、Scorpion-ARMS法により行った。上記操作により、EGFR exon18、19、20、21変異または欠損検体の同定を行った。
(Identification of EGFR gene mutation sample)
From a formalin-fixed paraffin-embedded tissue block in which tumor cells were confirmed by a HE-stained specimen, an unstained specimen slide was prepared in 10 μm-thick continuous sections and stored at room temperature. The sample was deparaffinized, and then the DNA in the sample was extracted using a Cobas DNA preparation kit. The detection of the EGFR gene mutation was performed by the Scorpion-ARMS method using a Cobas EGFR displacement detection kit. By the above operation, EGFR exon 18, 19, 20, 21 mutation or a defective specimen was identified.
 結果例
・図1の右図は感受性EGFR exon19欠損検体でありゲフェチニブに対する感受性が高いことが知られている(抗癌剤が効きやすい)
・図1の左図はWTでありゲフェチニブに対する感受性が低いことが知られている(抗癌剤が効かない)
 図1の右図のサンプルはゲフェチニブに感受性を示すようなEGF受容体の遺伝子変異が無く、本法によるゲフェチニブの感受性が弱いことが示された。一方、図1の左図のサンプルはゲフェチニブに感受性を示すことが判明しているEGFRコード遺伝子のエクソン19の塩基配列に欠損がみられるサンプルで、本法によるゲフェチニブの感受性も弱いことが示された。
Example of results-The right figure in Fig. 1 is a sensitive EGFR exon19-deficient specimen and is known to be highly sensitive to gefetinib (anticancer drugs are effective)
・ The left figure of FIG. 1 is WT and is known to have low sensitivity to gefetinib (anticancer drugs do not work)
The sample on the right side of FIG. 1 has no EGF receptor gene mutation that is sensitive to gefetinib, indicating that the sensitivity of gefetinib by this method is weak. On the other hand, the sample in the left figure of FIG. 1 is a sample in which the nucleotide sequence of exon 19 of the EGFR-coding gene, which has been found to be sensitive to gefetinib, is shown to be less sensitive to gefetinib by this method. It was.

Claims (14)

  1.  初代癌細胞のスフェロイド作製方法であって、
     全体積中少なくとも1体積%以上の血清を含む培地を用意する工程と、
     細胞との接着性を抑制する処理を施した細胞培養基材を用意する工程と、
     癌細胞を含む組織片から細胞を分取する工程と、
     前記培地中、前記細胞培養基材上に細胞を播種し、前記細胞を培養する工程と、を含むことを特徴とする初代癌細胞のスフェロイド作製方法。
    A method for producing spheroids of primary cancer cells,
    Providing a medium containing at least 1% by volume of serum in the total volume;
    A step of preparing a cell culture substrate subjected to a treatment for suppressing adhesion to cells;
    Separating cells from a tissue fragment containing cancer cells;
    Seeding cells on the cell culture substrate in the medium, and culturing the cells. A method for producing spheroids of primary cancer cells, comprising the steps of:
  2.  前記培地が、インスリンを含む、請求項1に記載のスフェロイド作製方法。 The spheroid production method according to claim 1, wherein the culture medium contains insulin.
  3.  前記インスリンの濃度が、培地中1μg/mL~100μg/mLである、請求項2に記載のスフェロイド作製方法。 The method for producing spheroids according to claim 2, wherein the concentration of the insulin is 1 μg / mL to 100 μg / mL in the medium.
  4.  前記培地が、ヒドロコルチゾン及び上皮成長因子から選ばれる1種以上をさらに含む、請求項1~3いずれか1項に記載のスフェロイド作製方法。 The spheroid production method according to any one of claims 1 to 3, wherein the medium further contains one or more selected from hydrocortisone and epidermal growth factor.
  5.  前記培地が、トランスフェリン、セレン酸ナトリウム、亜セレン酸ナトリウム、ピルビン酸ナトリウム、及びグルタミンから選ばれる1種以上をさらに含む、請求項1~4いずれか1項に記載のスフェロイド作製方法。 The spheroid production method according to any one of claims 1 to 4, wherein the medium further contains at least one selected from transferrin, sodium selenate, sodium selenite, sodium pyruvate, and glutamine.
  6.  前記培地が、エタノールアミン、トリヨードサイロニン、BSA、及びフォスフォリルエタノールアミンから選ばれる1種以上をさらに含む、請求項1~5いずれか1項に記載のスフェロイド作製方法。 The spheroid production method according to any one of claims 1 to 5, wherein the medium further contains one or more selected from ethanolamine, triiodothyronine, BSA, and phosphorylethanolamine.
  7.  前記細胞培養基材が、凸部上面が細胞接着面として機能する所定の凹凸構造を有する、請求項1~6いずれか1項に記載のスフェロイド作製方法。 The method for producing a spheroid according to any one of claims 1 to 6, wherein the cell culture substrate has a predetermined concavo-convex structure in which a convex upper surface functions as a cell adhesion surface.
  8.  前記凹凸構造の凸部上面がピラー状、ドット状、ライン状、複数の連続したまたは連続しない多角形のパターン構造を有する、請求項7に記載のスフェロイド作製方法。 The method for producing a spheroid according to claim 7, wherein the upper surface of the convex portion of the concave-convex structure has a pillar shape, a dot shape, a line shape, or a plurality of continuous or non-continuous polygon pattern structures.
  9.  前記多角形のパターン構造間の幅が20μm以下である、請求項8に記載のスフェロイド作製方法。 The method for producing a spheroid according to claim 8, wherein a width between the polygonal pattern structures is 20 µm or less.
  10.  請求項1~9いずれか1項に記載のスフェロイド作製方法により得たことを特徴とする、スフェロイド。 A spheroid obtained by the spheroid production method according to any one of claims 1 to 9.
  11.  請求項10に記載のスフェロイドを得る工程と、
     前記スフェロイドに対して被験物質を投与する工程と、を含むことを特徴とする
     スフェロイドに直接的又は間接的に作用を及ぼす物質のスクリーニング方法。
    Obtaining a spheroid according to claim 10;
    Administering a test substance to the spheroid, and a screening method for a substance that acts directly or indirectly on the spheroid.
  12.  請求項10に記載のスフェロイドを得る工程と、
     前記スフェロイドに対して薬剤を投与し、薬剤による反応を観察する工程と、を含むことを特徴とする
     スフェロイドに対する前記薬剤の感受性の判定方法。
    Obtaining a spheroid according to claim 10;
    Administering a drug to the spheroid and observing a reaction by the drug, and a method for determining the sensitivity of the drug to the spheroid.
  13.  請求項10に記載のスフェロイドを得る工程と、
     前記スフェロイドに対して薬剤を投与し、薬剤による反応を観察する工程と、
     がん細胞を含む組織片から分取した細胞の遺伝子配列を分析する工程と、
     薬剤による反応と遺伝子配列分析結果を照合する工程と、を含むことを特徴とする
     スフェロイドに対する前記薬剤の感受性の判定方法。
    Obtaining a spheroid according to claim 10;
    Administering a drug to the spheroid and observing a reaction due to the drug;
    Analyzing the gene sequence of cells taken from a tissue fragment containing cancer cells;
    A method for determining the sensitivity of the drug to spheroids, comprising the step of collating a reaction by the drug with a result of gene sequence analysis.
  14.  初代癌細胞のスフェロイドを作製するためのキットであって、
     全体積中少なくとも1体積%以上の血清を含む培地と、
     細胞との接着性を抑制する処理を施した細胞培養基材とを備える、キット。
    A kit for producing spheroids of primary cancer cells,
    A medium containing at least 1% by volume of serum in the total volume;
    A kit comprising a cell culture substrate subjected to a treatment for suppressing adhesion to cells.
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