WO2016047801A1 - Procédé de fabrication de sphéroïde de cellule cancéreuse initiale, sphéroïde, procédé de criblage, et procédé de jugement - Google Patents

Procédé de fabrication de sphéroïde de cellule cancéreuse initiale, sphéroïde, procédé de criblage, et procédé de jugement 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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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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Publication of WO2016047801A1 publication Critical patent/WO2016047801A1/fr

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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0693—Tumour cells; Cancer cells
    • G—PHYSICS
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5082—Supracellular entities, e.g. tissue, organisms
    • G01N33/5085—Supracellular entities, e.g. tissue, organisms of invertebrates
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; 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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    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/05—Inorganic components
    • C12N2500/10—Metals; Metal chelators
    • C12N2500/20—Transition metals
    • C12N2500/24—Iron; Fe chelators; Transferrin
    • C12N2500/25—Insulin-transferrin; Insulin-transferrin-selenium
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    • C12N2500/00—Specific components of cell culture medium
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
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    • C12N2501/11—Epidermal growth factor [EGF]
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/30—Hormones
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    • C12N2501/39—Steroid hormones
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
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    • C12N2501/395—Thyroid hormones
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    • C12N2503/00—Use of cells in diagnostics
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    • C12N2513/00—3D culture
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    • C12N2535/00—Supports or coatings for cell culture characterised by topography
    • C12N2535/10—Patterned coating
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/10—Screening 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

L'invention fournit un environnement de culture inhibant la capacité de prolifération de cellules dont la prolifération est rapide dans des tissus contaminés par de telles cellules, et avantageux pour une culture de cellules cancéreuses. L'invention fournit également un procédé de fabrication de sphéroïde permettant d'obtenir un sphéroïde contenant une cellule dérivée d'une cellule cancéreuse initiale en tant que composant principal. Ce procédé de fabrication de sphéroïde de cellule cancéreuse initiale est caractéristique en ce qu'il inclut : une étape au cours de laquelle est préparé un milieu de culture contenant un sérum à raison d'au moins 1% en volume de l'ensemble du produit; une étape une étape au cours de laquelle est préparé un matériau de base de culture cellulaire soumis à un traitement inhibant l'adhérence aux cellules; une étape au cours de laquelle sont fractionnées des cellules provenant d'un fragment de tissus contenant des cellules cancéreuses; et une étape au cours de laquelle des cellules sont implantées sur ledit matériau de base de culture cellulaire et mises en cultures dans ledit milieu de culture.
PCT/JP2015/077297 2014-09-26 2015-09-28 Procédé de fabrication de sphéroïde de cellule cancéreuse initiale, sphéroïde, procédé de criblage, et procédé de jugement WO2016047801A1 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2018011576A (ja) * 2016-07-22 2018-01-25 地方独立行政法人 大阪府立病院機構 初代細胞培養法
WO2018134778A1 (fr) * 2017-01-19 2018-07-26 University Of Cape Town Propagation in vitro de cellules cancéreuses primaires
WO2018169007A1 (fr) 2017-03-16 2018-09-20 株式会社Ｌｓｉメディエンス Culture tridimensionnelle de cellules cancéreuses primaires utilisant un tissu tumoral
CN111670247A (zh) * 2017-12-08 2020-09-15 京诊断株式会社 制备癌球状体的方法和选择结直肠癌患者的方法

Citations (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2005198659A (ja) * 1992-05-13 2005-07-28 Univ Texas Syst 検出方法
JP2011024580A (ja) * 2002-07-17 2011-02-10 Max-Planck-Ges Zur Foerderung Der Wissenschaften Ev 癌細胞浸潤の診断及び予防
WO2011149013A1 (fr) * 2010-05-26 2011-12-01 株式会社Ｒｅｉメディカル Procédé pour évaluer la sensibilité d'une masse cellulaire dérivée de tissus cancéreux ou d'une masse cellulaire cancéreuse agrégée à un agent médical ou à un rayonnement radioactif
JP2012187097A (ja) * 2010-12-13 2012-10-04 Shiseido Co Ltd 細胞凝集塊の形成方法
WO2013042360A1 (fr) * 2011-09-20 2013-03-28 株式会社クラレ Procédé de culture de cellules adhérentes
JP2013208104A (ja) * 2012-03-02 2013-10-10 Yamaguchi Univ 消化器系がん幹細胞を培養するための無血清培地、及びそれを用いた消化器系がん幹細胞の増殖方法
WO2014148562A1 (fr) * 2013-03-19 2014-09-25 国立大学法人 岡山大学 Méthode permettant d'obtenir une masse cellulaire contenant une cellule souche cancéreuse
JP2015047085A (ja) * 2013-08-30 2015-03-16 日油株式会社 細胞凝集塊の製造方法、及び薬剤のスクリーニング方法
JP2015047083A (ja) * 2013-08-30 2015-03-16 日油株式会社 細胞凝集塊の製造方法、及び薬剤のスクリーニング方法
JP2015047084A (ja) * 2013-08-30 2015-03-16 日油株式会社 細胞凝集塊の製造方法、及び薬剤のスクリーニング方法
JP2015062400A (ja) * 2013-08-30 2015-04-09 独立行政法人放射線医学総合研究所 癌組織由来細胞凝集塊を調製するための方法及び癌組織由来細胞凝集塊を用いる抗癌剤スクリーニング方法、抗癌剤の定量分析又は癌組織の放射線感受性試験

Patent Citations (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2005198659A (ja) * 1992-05-13 2005-07-28 Univ Texas Syst 検出方法
JP2011024580A (ja) * 2002-07-17 2011-02-10 Max-Planck-Ges Zur Foerderung Der Wissenschaften Ev 癌細胞浸潤の診断及び予防
WO2011149013A1 (fr) * 2010-05-26 2011-12-01 株式会社Ｒｅｉメディカル Procédé pour évaluer la sensibilité d'une masse cellulaire dérivée de tissus cancéreux ou d'une masse cellulaire cancéreuse agrégée à un agent médical ou à un rayonnement radioactif
JP2012187097A (ja) * 2010-12-13 2012-10-04 Shiseido Co Ltd 細胞凝集塊の形成方法
WO2013042360A1 (fr) * 2011-09-20 2013-03-28 株式会社クラレ Procédé de culture de cellules adhérentes
JP2013208104A (ja) * 2012-03-02 2013-10-10 Yamaguchi Univ 消化器系がん幹細胞を培養するための無血清培地、及びそれを用いた消化器系がん幹細胞の増殖方法
WO2014148562A1 (fr) * 2013-03-19 2014-09-25 国立大学法人 岡山大学 Méthode permettant d'obtenir une masse cellulaire contenant une cellule souche cancéreuse
JP2015047085A (ja) * 2013-08-30 2015-03-16 日油株式会社 細胞凝集塊の製造方法、及び薬剤のスクリーニング方法
JP2015047083A (ja) * 2013-08-30 2015-03-16 日油株式会社 細胞凝集塊の製造方法、及び薬剤のスクリーニング方法
JP2015047084A (ja) * 2013-08-30 2015-03-16 日油株式会社 細胞凝集塊の製造方法、及び薬剤のスクリーニング方法
JP2015062400A (ja) * 2013-08-30 2015-04-09 独立行政法人放射線医学総合研究所 癌組織由来細胞凝集塊を調製するための方法及び癌組織由来細胞凝集塊を用いる抗癌剤スクリーニング方法、抗癌剤の定量分析又は癌組織の放射線感受性試験

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
GOJI, H. ET AL.: "Abstract 329: Establishment of three-dimensional primary tumor cell culture method and novel drug sensitivity test", CANCER RESEARCH, vol. 75, no. 15 Suppl, ISSN: 1538-7445 *
INGRAM, P. ET AL.: "Spheroid cell culture on PDMS hydrophobic surfaces and integration into microfludic devices", INTERNATIONAL CONFERENCE ON MINIATURIZED SYSTEMS FOR CHEMISTRY AND LIFE SCIENCES, 2011, pages 1539 - 1541 *
LIU, T. ET AL.: "Quasi-spherical microwells on superhydrophobic substrates for long term culture of multicellular spheroids and high throughput assays", BIOMATERIALS, vol. 35, pages 6060 - 6068, XP028664980, ISSN: 0142-9612, doi:10.1016/j.biomaterials.2014.04.047 *
MASARU TAKAHASHI: "Efficacy of a Serum- Freemedium, RITC 80-7, for the Establishment of Human Cancer Cell Lines and Characterization of Established Cell Lines of KK-1 and GGK-1", SUMMARY OF THESIS, 1987, Zairyo Oyobi Hoho, Retrieved from the Internet <URL:http://ir.library.tohoku.ac.jp/re/bitstream/10097/19817/1/M1S620973.pdf> *

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JP2018011576A (ja) * 2016-07-22 2018-01-25 地方独立行政法人 大阪府立病院機構 初代細胞培養法
US10683485B2 (en) 2016-07-22 2020-06-16 Osaka Prefectural Hospital Organization Method for culturing primary cells
GB2572914A (en) * 2017-01-19 2019-10-16 Univ Cape Town In vitro propagation of primary cancer cells
WO2018134778A1 (fr) * 2017-01-19 2018-07-26 University Of Cape Town Propagation in vitro de cellules cancéreuses primaires
GB2572914B (en) * 2017-01-19 2022-11-23 Univ Cape Town In vitro propagation of primary cancer cells
WO2018169007A1 (fr) 2017-03-16 2018-09-20 株式会社Ｌｓｉメディエンス Culture tridimensionnelle de cellules cancéreuses primaires utilisant un tissu tumoral
CN110475860A (zh) * 2017-03-16 2019-11-19 美迪恩斯生命科技株式会社 使用肿瘤组织的原代癌细胞的三维培养
JPWO2018169007A1 (ja) * 2017-03-16 2020-02-13 株式会社Ｌｓｉメディエンス 腫瘍組織を用いた初代がん細胞の３次元培養
US11549100B2 (en) 2017-03-16 2023-01-10 Lsi Medience Corporation Three-dimensional culture of primary cancer cells using tumor tissue
US11873514B2 (en) 2017-03-16 2024-01-16 Lsi Medience Corporation Method of screening for a substance that acts on a cell mass
CN110475860B (zh) * 2017-03-16 2024-05-14 美迪恩斯生命科技株式会社 使用肿瘤组织的原代癌细胞的三维培养
CN111670247A (zh) * 2017-12-08 2020-09-15 京诊断株式会社 制备癌球状体的方法和选择结直肠癌患者的方法
CN111670247B (zh) * 2017-12-08 2024-01-05 京诊断株式会社 制备癌球状体的方法和选择结直肠癌患者的方法

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