WO2011077894A1 - Method for enhancing formation of spheroid and culture medium for enhancing formation of spheroid - Google Patents

Method for enhancing formation of spheroid and culture medium for enhancing formation of spheroid Download PDF

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WO2011077894A1
WO2011077894A1 PCT/JP2010/071002 JP2010071002W WO2011077894A1 WO 2011077894 A1 WO2011077894 A1 WO 2011077894A1 JP 2010071002 W JP2010071002 W JP 2010071002W WO 2011077894 A1 WO2011077894 A1 WO 2011077894A1
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medium
cells
extracellular matrix
spheroid formation
promoting
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PCT/JP2010/071002
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French (fr)
Japanese (ja)
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小林真喜
吉田かおり
脇厚生
田中覚
吉井幸恵
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Ｓｃｉｖａｘ株式会社
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0062—General methods for three-dimensional culture
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/90—Substrates of biological origin, e.g. extracellular matrix, decellularised tissue

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  • the present invention relates to a culture method for promoting spheroid formation and a cell culture medium for promoting spheroid formation.
  • Spheroid means an aggregate of cells in which cells are aggregated and aggregated three-dimensionally. It is known that spheroids can maintain a specific function of cells for a long time as compared with cells by monolayer culture. Therefore, in recent years, spheroids are used as tools for pharmacological activity evaluation, toxicity tests, and functional analysis in drug development and food development.
  • Examples of the method for forming spheroids include a method of forming spheroids in a state where cells are suspended in a funnel-shaped well having a surface with low cell adhesion (for example, see Patent Document 1), or a predetermined uneven structure surface. There is a method of forming spheroids in a state in which cells are adhered (see, for example, Patent Document 2 and Patent Document 3).
  • an object of the present invention is to provide a cell culture method that can form spheroids easily and in a short period of time, and a spheroid formation promoting medium that can be used in the method.
  • the present inventors have conducted intensive studies. As a result, a single layer in which spheroids were not formed by culturing cells using a cell culture medium containing a predetermined extracellular matrix on a culture substrate having a predetermined concavo-convex structure that functions as a cell adhesion surface It was found that the proliferation and growth of cells and the separation and collapse of formed spheroids were prevented, and the formation of spheroids was promoted extremely effectively, and the present invention was completed.
  • the gist of the present invention is as follows.
  • the concavo-convex structure is formed by regularly arranging a plurality of unit structures having a width between unit structures of 3 ⁇ m or less, a planar shape of a polygon and a minimum inner diameter of 3 ⁇ m or less. The method for promoting spheroid formation according to (1).
  • Method. The method for promoting spheroid formation according to any one of (1) to (5), wherein the extracellular matrix component is an extract from an Engelbreth-Holm-Swarm (EHS) mouse tumor.
  • EHS Engelbreth-Holm-Swarm
  • (10) Used for cells on the concavo-convex structure formed by regularly arranging a plurality of unit structures each having a width between unit structures of 3 ⁇ m or less and a planar shape of a polygon and a minimum inner diameter of 3 ⁇ m or less.
  • the medium for promoting spheroid formation according to (9), wherein (11) The spheroid formation promoting medium according to any one of (9) and (10), wherein the content of the extracellular matrix component is 0.05 mg / ml or less. (12) The spheroid formation promoting medium according to any one of (9) and (10), wherein the content of the extracellular matrix component is 0.02 mg / ml or less.
  • the spheroid formation promoting medium according to any one of (9) to (13), wherein the extracellular matrix component is an extract from an Engelbreth-Holm-Swarm (EHS) mouse tumor.
  • EHS Engelbreth-Holm-Swarm
  • spheroids can be formed according to the present invention, for example, in drug screening and toxicity evaluation, it is possible to reduce noise caused by mixing of monolayer cells, separated spheroids, and the like. Moreover, since spheroids can be formed in a short period of time, it is possible to use spheroids that save cost and labor.
  • FIG. 3 is a photograph of NCI-H2030 cells cultured in a medium with a Matrigel concentration of 0 to 0.05 mg / ml.
  • 2 is a photograph of HuH-7 cells cultured in a medium with a Matrigel concentration of 0 to 0.01 mg / ml.
  • 2 is a photograph of Hs578T cells cultured in a medium with a Matrigel concentration of 0 to 0.05 mg / ml.
  • 2 is a photograph of A549 cells cultured in a medium having a matrigel concentration of 0 to 0.05 mg / ml.
  • FIG. 4 is a photograph of PC-3 cells cultured in NCM medium and Ham's F-12K medium having a Matrigel concentration of 0 to 0.05 mg / ml.
  • 2 is a photograph of Panc-1 cells cultured in NCM medium and RPMI medium having a Matrigel concentration of 0 to 0.05 mg / ml.
  • 2 is a photograph of BxPC-3 cells cultured in NCM medium and RPMI medium having a Matrigel concentration of 0 to 0.05 mg / ml. It is a photograph of colo205 cells cultured in NCM medium and RPMI medium having a Matrigel concentration of 0 to 0.05 mg / ml.
  • FIG. 2 is a photograph of MDA-MB-231 cells cultured in NCM medium and L-15 medium having a matrigel concentration of 0 to 0.05 mg / ml. It is a photograph of Panc-1 cells cultured in a medium supplemented with Matrigel components. It is a photograph of BxPC-3 cells cultured in a medium supplemented with Matrigel components. The It is a photograph of colo205 cells cultured in a medium supplemented with Matrigel components.
  • the Spheroid Formation Promotion Method of the Present Invention is characterized in that cells are cultured in the presence of a basic medium component and an extracellular matrix on a culture substrate having an uneven structure that functions as a cell adhesion surface. This is a method for promoting the formation of spheroids.
  • the spheroid here refers to an aggregate of cells in which cells are aggregated and aggregated three-dimensionally.
  • the shape of the concavo-convex structure functioning as a cell adhesion surface in the present invention can be various, such as linear (line and space), pillar, hole, etc., depending on the difference in adhesion of the cells that form spheroids to the culture substrate.
  • a structure in which a plurality of unit structures (polygons such as triangles, quadrilaterals, hexagons, circles, and other shapes) having a predetermined planar shape are regularly arranged.
  • a plurality of unit structures 1 having a polygonal planar shape can be formed as a continuous structure.
  • regular polygons such as regular triangles, squares, regular hexagons, and circles are more preferable in that spheroids can be grown on an isotropically uniform structure.
  • the width between unit structures is preferably as small as 3 ⁇ m or less, 2 ⁇ m or less, 1 ⁇ m or less, 700 nm or less, 500 nm or less, or 250 nm or less. This is because the smaller the width between the unit structures, the more the cells adhered to the concavo-convex structure surface are considered to be able to form spheroids while growing many pseudopods.
  • the depth of the unit structure is 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 depending on the difference in adhesion of the cells for forming spheroids to the culture substrate. It is formed in various sizes as described above. Further, the aspect ratio of the unevenness includes various ones such as 0.2 or more, 0.5 or more, 1 or more, 2 or more.
  • the minimum inner diameter (preferably the maximum inner diameter) of the unit structure is preferably 3 ⁇ m or less, and it is preferable for the same reason as described above to be smaller, such as 2 ⁇ m or less, 1 ⁇ m or less, 700 nm or less, 500 nm or less, or 250 nm or less.
  • the inner diameter means the distance between two parallel lines circumscribing the unit structure
  • the minimum inner diameter means the shortest distance among the two parallel lines circumscribing the unit structure
  • the maximum inner diameter means the longest distance between two parallel lines circumscribing the unit structure.
  • the unit structure when the unit structure is a regular hexagon, the distance between the parallel sides facing each other is the minimum inner diameter, and the distance between the opposite vertices is the maximum inner diameter.
  • the unit structure when the unit structure is rectangular, the length of the short side is the minimum inner diameter, and the length of the diagonal line is the maximum inner diameter.
  • NanoCulture plate (registered trademark: SCIVAX Co., Ltd.) can be mentioned.
  • the shape of the culture substrate used in the present invention may be any shape as long as cells can be cultured.
  • it can be formed into a film shape or a substrate shape (plate shape), such as a petri dish, a dish, It can be used for multiwell plates, flasks, chamber slides and the like.
  • the uneven structure should just be formed in at least one part on a base material.
  • the material of the culture substrate may be any material as long as it is non-toxic to cells.
  • polystyrene polyethylene”, “polypropylene”, “polyimide”, “polylactic acid or polylactic acid” -Biodegradable polymers such as polyglycolic acid copolymer, polycaprolactone "," cyclic olefin thermoplastic resins such as cyclic olefin copolymer (COC) and cyclic olefin polymer (COP) ",” acrylic resin “, “Other resins such as photo-curing resins and thermosetting resins”, “metals such as aluminum oxide”, “glass”, “quartz glass”, “silicon”, etc. can be used, and are made of silicon, glass, etc.
  • a substrate in which a coating layer such as “resin”, “photoresist”, or “metal such as aluminum oxide” is formed on the surface of the substrate body can also be used.
  • the surface of the culture substrate may be one whose hydrophilicity is controlled.
  • the control method include, but are not limited to, surface modification technology by irradiation with ultraviolet rays, electron beams, gamma rays, plasma, etc., coating with silicon dioxide (SiO 2 ), polylysine, various extracellular matrices, and the like. It is not a thing.
  • the cell culture substrate can be produced by any method as long as it can form a concavo-convex structure.
  • nanoimprint technology for example, nanoimprint technology, solution casting method, etching, blasting, corona discharge and 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.
  • the medium basic component is composed of a carbon source that can be assimilated by cells, a nitrogen source that can be digested, and an inorganic salt, and specifically includes, for example, inorganic salts, amino acids, carbohydrates, and vitamins. Is included.
  • Examples of minerals include, but are not limited to, calcium, potassium, magnesium, phosphorus, sodium, copper, iron, selenium, manganese, silicon, molybdenum, vanadium, nickel, and zinc.
  • amino acids examples include L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine, L-glutamic acid, L-glutamine, glycine, L-histidine, L-isoleucine, L-leucine, L Examples include, but are not limited to, lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, and L-valine.
  • carbohydrates include, but are not limited to, glucose, galactose, mannose, fructose and the like.
  • vitamins examples include D-biotin, folinic acid, DL- ⁇ -lipoic acid, nicotinamide, D-pantothenic acid, pyridoxine, riboflavin, thiamine, cyanocobalamin (vitamin B 12 ), etc. It is not limited to.
  • basal medium containing the basal medium examples include eagle medium (for example, BME, MEM, DMEM, GMEM), RPMI medium, L-15 medium, Fisher medium, ham medium (for example, F10, F12), MCDB medium, and the like. However, it is not limited to these.
  • the extracellular matrix component is a substance that surrounds cells in the living body, and is a substance that plays a skeletal role, a role of a scaffold in cell adhesion, a role of holding / providing a cell growth factor, or the like. It refers to a substance that exhibits similar biological activity.
  • extracellular matrix components used in the method for promoting spheroid formation of the present invention include fibrous proteins such as collagen, elastin, and fibrillin, and glycosami such as hyaluronic acid, chondroitin sulfate, dermatan sulfate, heparan sulfate, heparin, and ketalan sulfate.
  • Cell adhesion proteins such as noglycan and proteoglycan, fibronectin, laminin, vitronectin, tenascin, thrombospondin, entactin, nidogen, osteopontin, von Willebrand factor, fibrinogen, etc. It is not particularly limited.
  • the extracellular matrix may be artificially made. In the present invention, these may be used alone or in combination.
  • a basement membrane component can be used as an extracellular matrix component used in the spheroid formation promoting method of the present invention.
  • the basement membrane is known to play an important role in various processes including cell adhesion, motility, proliferation, functional maintenance, and differentiation.
  • Typical basement membrane components include laminin, type IV collagen, entactin, heparin, and heparan sulfate proteoglycan.
  • an extract from an animal or a preparation thereof can be used.
  • an extract from an Engelbreth-Holm-Swarm (EHS) mouse tumor is used.
  • EHS Engelbreth-Holm-Swarm
  • Examples of commercially available products prepared from the extract include “Matrigel” (registered trademark: BD Bioscience) and ECM gel (Sigma-Aldrich).
  • the composition and content of the components contained in the preparation are not particularly limited, but when used for research purposes such as signal research, growth factor function elucidation, gene expression research, drug screening, etc. It is desirable that the growth factor content is reduced.
  • laminin, type IV collagen, laminin / entactin complex which is a constituent component of the “Matrigel”
  • laminin, type IV collagen, laminin / entactin complex which is a constituent component of the “Matrigel”
  • the extracellular matrix component used in the method for promoting spheroid formation of the present invention laminin, type IV collagen, laminin / entactin complex, which is a constituent component of the “Matrigel”
  • the extracellular matrix component used in the method for promoting spheroid formation of the present invention laminin, type IV collagen, laminin / entactin complex, which is a constituent component of the “Matrigel”
  • the disadvantage of “Matrigel” that a stable result cannot be obtained due to a large lot difference can be solved.
  • the content of the extracellular matrix component in the medium is a concentration that does not gel at the culture temperature during spheroid formation, the type of cells to be cultured, the type of basal medium component, etc. It can be appropriately changed depending on the situation.
  • the concentration that does not cause gelation refers to a concentration that does not affect handling such as medium exchange, various assays, cell recovery, and the like, and a concentration that can be smoothly transferred to an evaluation system after culture. Specifically, it is 10% or less, for example.
  • the content of the extracellular matrix component effective for promoting spheroid formation in the medium is, for example, 0.05 mg / ml or less, preferably 0.02 mg / ml or less, 0.01 mg / ml or less, 0. 005 mg / ml or less.
  • the spheroid formation promotion method of the present invention further includes serum from bovine, horse, human, etc., and various growth factors (for example, EGF, bFGF, NGF, PDGF, IGF-1, TGF- ⁇ , VEGF, TNF- ⁇ ), substances known to be effective in cell growth and maintenance (eg, serum albumin, transferrin, lipid, lipid acid source, cholesterol, pyruvate, glucocorticoid, DNA And RNA synthesis nucleosides), trace metal elements, surfactants, cytokines, differentiation-inducing factors, and the like.
  • growth factors for example, EGF, bFGF, NGF, PDGF, IGF-1, TGF- ⁇ , VEGF, TNF- ⁇
  • substances known to be effective in cell growth and maintenance eg, serum albumin, transferrin, lipid, lipid acid source, cholesterol, pyruvate, glucocorticoid, DNA And RNA synthesis nucleosides
  • the spheroid formation rate varies depending on the cell type. This is considered to be due to the difference in the adhesion of the cells to the culture substrate. Therefore, in order to control the adhesion of cells to the culture substrate, depending on the cell type, the planar shape of the concavo-convex structure, the width between unit structures, the material of the culture substrate, the hydrophilicity, etc. and the basis of the medium used It is possible to improve the rate of spheroid formation by adjusting the medium components.
  • a predetermined amount of extracellular matrix components contained in the medium provide physical support to the cells, and biochemical stimulation via receptors on the cell surface and the like. It seems to enhance cell-cell adhesion. For this reason, it is considered that a change occurs in the balance between the adhesion between cells and the adhesion between the cells and the culture substrate, and the formation of spheroids is promoted.
  • the cell used in the present invention may be any cell as long as it can form a spheroid, but is preferably an animal-derived cell, and more preferably a mammal-derived cell.
  • mammals include, but are not limited to, humans, dogs, cats, monkeys, cows, pigs, sheep, horses, rats, mice and the like.
  • the cells used in the present invention include mesoderm tissue cells including hematopoietic cells and mesenchymal cells, endoderm tissue cells, ectoderm tissue cells or any cells included in the process of differentiation from fertilized eggs to these cells, and Any of stem cells such as embryonic stem cells may be used.
  • stem cells such as embryonic stem cells may be used.
  • hematopoietic cells include hematopoietic stem cells, hematopoietic progenitor cells, red blood cells, lymphocyte cells, granulocyte cells, and platelet cells.
  • Mesenchymal cells are cells of connective tissues such as bone cells, chondrocytes, muscle cells, tendon cells, adipocytes, dermal papilla cells, pulp cells, and the ability to differentiate into these cells.
  • Such cells may be established as cultured cells or primary cells obtained from biological tissues. Further, for example, it may be a tumor cell, a cell transformed by a genetic engineering technique, a cell infected by a viral vector, or the like.
  • epithelial cells such as primary hepatocytes, hepatocyte cell lines, primary cancer cells and cancer cell lines are preferred, such as A549 cells (human lung cancer cell lines), HuH-7 cells (human hepatoma cell lines).
  • PC-3 cells human prostate cancer cell line
  • NCI-H2030 cells human lung adenocarcinoma cell line
  • Hs578T cells human breast cancer cell line
  • Panc-1 cells human pancreatic cancer cell line
  • BxPC-3 Suitable cells include cells (human pancreatic cancer cell line), colo205 cells (human colon cancer cell line), MDA-MB-231 cells (human breast cancer cell line) and the like.
  • the method for allowing the medium basic component and the extracellular matrix component to coexist is not particularly limited, and the extracellular matrix component may be added to the medium basic component, and the medium basic component may be added to the container. And extracellular matrix components may coexist simultaneously.
  • a medium containing a medium basic component and a predetermined extracellular matrix component can be used.
  • the medium basic component and / or the extracellular matrix component and the cell can coexist with, for example, a method of adding cells to a medium containing the medium basic component and extracellular matrix component, or a cell in the medium containing the basic medium component. Examples thereof include a method of adding an extracellular matrix component during the culture.
  • the timing at which the medium basic component and extracellular matrix component coexist with the cells is not particularly limited.
  • the entire period from the start to the end of the culture or a part of the period may be used. In order to maintain the ability to form spheroids, it is more convenient to use the former period.
  • Culture conditions are not particularly limited as long as they follow conventional methods.
  • the oxygen concentration, osmotic pressure, pH, medium temperature, and the like in the medium can be appropriately changed according to the type of cells to be cultured, the purpose of culture, the culture amount, the type of basal medium components, and the like.
  • it may be cultured for 3-14 days at about 37 ° C. in an atmosphere where the gas phase CO 2 concentration is 0-40%.
  • the spheroid formation promoting medium of the present invention is used for a culture substrate having a concavo-convex structure that functions as a cell adhesion surface, and contains a medium basic component and a predetermined extracellular matrix. A medium for promoting the formation of spheroids.
  • the culture substrate and the production method thereof For the shape of the concavo-convex structure in the spheroid formation promoting medium of the present invention, the same products and methods as described above can be used.
  • the same materials as described above can be used.
  • Matrigel (registered trademark, BD Bioscience, BD Matrigel Growth Factor Reduce) was added to a medium (SCVAX NCM-M) to a concentration of 0 to 5.0%, and NCI-H2030 cells (CRL-) were added in the medium.
  • SCVAX NCM-M serum-free medium
  • NCI-H2030 cells CCL-
  • 5914: Human lung adenocarcinoma cell line was cultured for 7 days. Culturing was performed by seeding NCI-H2030 cells at 1 ⁇ 10 4 cells / 100 ⁇ l / well in a 96-well plate under conditions of 37 ° C. and 5% CO 2 .
  • Spheroids were hardly formed in the medium to which no Matrigel was added (Matrigel concentration 0 vol%).
  • small spheroids were formed in the medium to which Matrigel was added in an amount of 0.1 to 1.0 vol%, and the number thereof increased depending on the concentration.
  • large spheroids were formed at a Matrigel concentration of 2.0% to 5.0 vol%.
  • Matrigel (registered trademark BD Bioscience BD Matrigel Growth Factor Reduced) was added to a medium (SCIVAX NCM-M) to a concentration of 0 to 1.0 vol%, and HuH-7 cells (RCB1366: A cultured cell line derived from human liver cancer) was cultured for 7 days. The culture was performed by seeding HuH-7 cells in 96-well plates at 1 ⁇ 10 4 cells / 100 ⁇ l / well at 37 ° C. under 5% CO 2 conditions.
  • Spheroids are hardly formed in the medium to which Matrigel is not added (Matrigel concentration 0 vol%), and cells that grow and proliferate in two dimensions in addition to cells that form spheroids at a matrigel concentration of 0.2% to 0.5 vol%. Were mixed. In contrast, when the Matrigel concentration was 1.0 vol%, the number of cells that grew and proliferated in two dimensions decreased, and both the number and size of spheroids were improved.
  • Hs578T cells (HTB-126: Human breast cancer cell line) was cultured for 7 days. Culturing was performed by seeding Hs578T cells at 1 ⁇ 10 4 cells / 100 ⁇ l / well in a 96-well plate at 37 ° C. and 5% CO 2 .
  • Spheroids were formed even in a medium without addition of Matrigel (Matrigel concentration 0 vol%), but larger spheroids were formed at a Matrigel concentration of 1.0 vol% or more, particularly at a Matrigel concentration of 1.0% to 2.0 vol%.
  • Matrigel (registered trademark BD Bioscience BD Matrigel Growth Factor Reduced) was added to a medium (SCIVAX NCM-M) to a concentration of 0 to 5.0 vol%, and A549 cells (RCB0098: human lung cancer) were added to the medium.
  • the cultured cell line was cultured for 7 days. Cultivation was performed by seeding A549 cells at 1 ⁇ 10 4 cells / 100 ⁇ l / well in a 96-well plate under conditions of 37 ° C. and 5% CO 2 .
  • spheroids were formed in the medium to which Matrigel was added, compared to the medium to which Matrigel was not added (Matrigel concentration 0 vol%). In particular, large spheroids were formed at a matrigel concentration of 1.0% to 5.0 vol%.
  • Matrigel (registered trademark BD Bioscience BD Matrigel Growth Factor Reduced) is added to a medium (SCIVAX NCM-M or Wako Ham's F12-K) to a concentration of 0 to 5.0 vol%, and PC-3 cells (CRL1435: cultured cell line derived from human prostate cancer) were cultured in the medium for 7 days. Cultivation was performed by seeding PC-3 cells in 96-well plates at 1 ⁇ 10 4 cells / 100 ⁇ l / well at 37 ° C. under 5% CO 2 conditions.
  • Spheroids were hardly formed in the medium to which no Matrigel was added (Matrigel concentration 0 vol%).
  • Matrigel concentration 0 vol%.
  • spheroids were formed at a Matrigel concentration of 1.0% to 5.0 vol%, and the best spheroids were formed particularly when Matrigel was 1.0 vol%.
  • spheroid-like cell clusters were also formed in Ham's F12-K medium supplemented with Matrigel.
  • Matrigel (registered trademark, BD Bioscience, BD Matrigel Growth Factor Reduced) is added to a medium (SCIVAX NCM-M or SIGMA RPMI1640) to a concentration of 0 to 5.0 vol%, and Panc- One cell (RCB2095: human pancreatic cancer cell line) was cultured for 7 days. Cultivation was performed by seeding Panc-1 cells in 96-well plates at 1 ⁇ 10 4 cells / 100 ⁇ l / well and under 37 ° C. and 5% CO 2 conditions.
  • spheroids are not formed, and spheroids are formed at a matrigel concentration of 1.0% to 5.0 vol%, and in particular, Matrigel 2.0% to 5.0 vol. %, Large spheroids were formed.
  • Matrigel (registered trademark BD Bioscience BD Matrigel Growth Factor Reduced) is added to a medium (SCIVAX NCM-M or SIGMA RPMI 1640) to a concentration of 0 to 5.0 vol%, and BxPC- 3 cells (CRL-1687: human pancreatic cancer cell line) was cultured for 7 days. Culturing was performed by seeding BxPC-3 cells at 1 ⁇ 10 4 cells / 100 ⁇ l / well in a 96-well plate at 37 ° C. and 5% CO 2 .
  • Matrigel (registered trademark, BD Bioscience, BD Matrigel Growth Factor Reduce) was added to a medium (SCIVAX NCM-M or SIGMA RPMI 1640) to a concentration of 0 to 5.0 vol%, and colo205 cells were added to the medium.
  • EC-87061208: Human colorectal cancer cell line was cultured for 7 days. Colo205 cells were seeded on a 96-well plate at 1 ⁇ 10 4 cells / 100 ⁇ l / well and cultured under conditions of 37 ° C. and 5% CO 2 .
  • NCM medium In both NCM medium and RPMI medium, spheroids are not formed when no matrigel is added (matrigel concentration 0 vol%), and spheroids are formed when the matrigel concentration is 0.5 to 5.0 vol%, and in particular, the matrigel concentration is 1.0 vol%. At that time, good spheroids were formed. NCM medium formed well-shaped spheroids, and RPMI medium formed large spheroids.
  • Matrigel (registered trademark BD Bioscience's BD Matrigel Growth Factor Reduce) medium (SCIVAX's NCM-M or SIGMA's L-15) at a concentration of 0 to 5.0 vol% MDA-MB-231 cells (HTB-26: human breast cancer cell line) was cultured for 7 days. Culturing was performed by seeding MDA-MB-231 cells in 96-well plates at 1 ⁇ 10 4 cells / 100 ⁇ l / well at 37 ° C. under 5% CO 2 conditions.
  • Matrigel registered trademark BD Bioscience manufactured by BD Matrigel Growth Factor Reduced Phenol Red Free
  • laminin Laminin manufactured by BD Bioscience
  • Laminin / entactin high concentration laminin / entactin complex (mouse) manufactured by BD Bioscience) was added to the culture medium (SCMAX NCM-M) at the concentration shown in Table 1, and Panc-1 Cells (RCB2095: human pancreatic cancer cell line) were cultured for 7 days. Cultivation was performed by seeding Panc-1 cells in 96-well plates at 1 ⁇ 10 4 cells / 100 ⁇ l / well and under 37 ° C.
  • Spheroids were hardly formed in the medium to which no Matrigel component was added (no addition), whereas small spheroids were formed in the medium to which type IV collagen or laminin or laminin / entactin complex was added. Large spheroids were formed in the medium supplemented with type IV collagen and laminin / entactin complex. This spheroid-forming ability was almost the same as when adding the same concentration of matrigel.
  • Matrigel registered trademark BD Bioscience manufactured by BD Matrigel Growth Factor Reduced Phenol Red Free
  • type IV collagen Collagen IV (mouse) manufactured by BD Bioscience
  • laminin Laminin manufactured by BD Bioscience
  • Laminin / entactin BD Bioscience's high-concentration laminin / entactin complex (mouse)
  • SCVAX NCM-M medium at the concentrations shown in Table 1
  • BxPC-3 was added in the medium.
  • Cells (CRL-1687: human pancreatic cancer cell line) was cultured for 7 days.
  • Small spheroids were formed in the medium not added with Matrigel component (no addition). In contrast, the number and size of spheroids improved in the medium supplemented with type IV collagen, laminin, or laminin / entactin complex. In the medium supplemented with type IV collagen and laminin / entactin complex, large spheroids were formed which were similar to those obtained when the same concentration of matrigel was added.
  • Matrigel registered trademark BD Bioscience manufactured by BD Matrigel Growth Factor Reduced Phenol Red Free
  • type IV collagen Collagen IV (mouse) manufactured by BD Bioscience
  • laminin Laminin manufactured by BD Bioscience
  • Laminin / entactin BD Bioscience's high-concentration laminin / entactin complex (mouse)
  • SCIVAX NCM-M colo205 cells
  • Colo205 cells were seeded on a 96-well plate at 1 ⁇ 10 4 cells / 100 ⁇ l / well and cultured under conditions of 37 ° C. and 5% CO 2 .
  • Spheroids were not formed in the medium to which no Matrigel component was added (no addition), whereas small spheroids were formed in the medium to which type IV collagen or laminin or laminin / entactin complex was added. In the medium supplemented with type IV collagen and laminin / entactin complex, the size of spheroids was further improved.
  • the present invention enables the formation of excellent spheroids, it can be used in a wide range of technologies related to the medical and biotechnology fields such as regenerative medicine, cell engineering, tissue engineering, pharmaceutical screening and toxicity evaluation, It is useful for evaluating the effectiveness and functionality of cosmetics and foods, and evaluating safety.

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Abstract

Disclosed is a cell cultivation method which is capable of forming a spheroid by simple processes in a short term. Also disclosed is a culture medium for enhancing the formation of a spheroid, which can be used for the cell cultivation method. By culturing cells on a culture substrate, which has a recessed and projected structure that functions as a cell adhesion surface, using a cell culture medium that contains a predetermined extracellular matrix component, the proliferation and growth of monolayer cells as well as separation and collapse of a spheroid are prevented, thereby extremely effectively enhancing the formation of the spheroid.

Description

スフェロイド形成促進方法およびスフェロイド形成促進培地Spheroid formation promotion method and spheroid formation promotion medium
　本発明は、スフェロイド形成を促進させる培養方法およびスフェロイド形成を促進させる細胞培養用培地に関する。 The present invention relates to a culture method for promoting spheroid formation and a cell culture medium for promoting spheroid formation.
　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。スフェロイドは単層培養による細胞と比較して、細胞の特異的な機能を長時間維持できることが知られている。そのため近年、スフェロイドは、薬剤開発や食品開発等において、薬理活性評価、毒性試験、機能解析のためのツールとして利用されている。 Spheroid means an aggregate of cells in which cells are aggregated and aggregated three-dimensionally. It is known that spheroids can maintain a specific function of cells for a long time as compared with cells by monolayer culture. Therefore, in recent years, spheroids are used as tools for pharmacological activity evaluation, toxicity tests, and functional analysis in drug development and food development.
　スフェロイドの形成方法としては、例えば、細胞接着性の低い表面を有するロート状のウェル中に細胞を浮遊させた状態でスフェロイドを形成させる方法（例えば、特許文献１参照）や、所定の凹凸構造面上に細胞を接着させた状態でスフェロイドを形成させる方法（例えば、特許文献２および特許文献３参照）がある。 Examples of the method for forming spheroids include a method of forming spheroids in a state where cells are suspended in a funnel-shaped well having a surface with low cell adhesion (for example, see Patent Document 1), or a predetermined uneven structure surface. There is a method of forming spheroids in a state in which cells are adhered (see, for example, Patent Document 2 and Patent Document 3).
特開平６－３２７４６２JP-A-6-327462 特開２００２－３３５９４９JP 2002-335949 A 特許４１５９１０３Patent 4159103
　しかしながら、従来技術では、スフェロイドの形成が難しい細胞種でスフェロイド培養を行うと、スフェロイド以外に単層細胞が混在してしまい、スフェロイドを利用した評価測定の精度が落ちる、さらには、評価系自体を構築することができないという問題があった。また、スフェロイド内の細胞同士の接着が弱いために、形成されたスフェロイドが分離して壊れやすいという問題があった。さらに、スフェロイドの形成に長期間を要するため、コストや手間がかかるという問題もあった。 However, in the prior art, when spheroid culture is performed with cell types in which spheroid formation is difficult, monolayer cells are mixed in addition to spheroids, and the accuracy of evaluation measurement using spheroids decreases. There was a problem that it could not be built. Moreover, since the adhesion | attachment of the cells in a spheroid is weak, there existed a problem that the formed spheroid isolate | separated and it was easy to break. Furthermore, since it takes a long time to form the spheroids, there is a problem that costs and labor are required.
　そこで、本発明は、簡便かつ短期間でスフェロイドを形成させることができる細胞培養方法および該方法に用いることのできるスフェロイド形成促進培地を提供することを目的とする。 Therefore, an object of the present invention is to provide a cell culture method that can form spheroids easily and in a short period of time, and a spheroid formation promoting medium that can be used in the method.
　上記目的を達成するために、本発明者らは鋭意検討を行った。その結果、細胞接着面として機能する所定の凹凸構造を有する培養基材上で、所定の細胞外マトリックスを含有する細胞用培地を用いて細胞を培養することにより、スフェロイドを形成しなかった単層細胞の増殖・成長や、形成されたスフェロイドの分離崩壊を防ぎ、極めて効果的にスフェロイドの形成が促進されることを見出し、本発明を完成させた。 In order to achieve the above object, the present inventors have conducted intensive studies. As a result, a single layer in which spheroids were not formed by culturing cells using a cell culture medium containing a predetermined extracellular matrix on a culture substrate having a predetermined concavo-convex structure that functions as a cell adhesion surface It was found that the proliferation and growth of cells and the separation and collapse of formed spheroids were prevented, and the formation of spheroids was promoted extremely effectively, and the present invention was completed.
　本発明の要旨は以下の通りである。 The gist of the present invention is as follows.
（１）　細胞接着面として機能する凹凸構造を有する培養基材上において、培地基礎成分と、培養温度にてゲル化しない濃度の細胞外マトリックス成分との存在下で、細胞を培養することを特徴とするスフェロイド形成促進方法。
（２）　前記凹凸構造は、単位構造間の幅が３μｍ以下で平面方向の形状が多角形であると共に最小内径が３μｍ以下の単位構造を規則的に複数配列して形成されていることを特徴とする（１）記載のスフェロイド形成促進方法。
（３）　前記細胞外マトリックス成分の含有量が０．０５ｍｇ／ｍｌ以下である（１）又は（２）いずれか記載のスフェロイド形成促進方法。
（４）　前記細胞外マトリックス成分の含有量が０．０２ｍｇ／ｍｌ以下である（１）又は（２）いずれか記載のスフェロイド形成促進方法。
（５）　前記細胞外マトリックス成分の含有量が０．０１ｍｇ／ｍｌ以下である（１）又は（２）いずれか記載のスフェロイド形成促進方法。
（６）　前記細胞外マトリックス成分が基底膜成分であることを特徴とする（１）乃至（５）いずれか記載のスフェロイド形成促進方法。
（７）　前記細胞外マトリックス成分が少なくともラミニン、ＩＶ型コラーゲン、ラミニン／エンタクチン複合体から選択される１以上の成分を含むことを特徴とする（１）乃至（５）いずれか記載のスフェロイド形成促進方法。
（８）　前記細胞外マトリックス成分がＥｎｇｅｌｂｒｅｔｈ－Ｈｏｌｍ－Ｓｗａｒｍ（ＥＨＳ）マウス腫瘍からの抽出物であることを特徴とする（１）乃至（５）いずれか記載のスフェロイド形成促進方法。
（９）　細胞接着面として機能する凹凸構造上の細胞に用いられるものであって、培地基礎成分と、培養温度にてゲル化しない濃度の細胞外マトリックス成分とを含有することを特徴とするスフェロイド形成促進培地。
（１０）　単位構造間の幅が３μｍ以下で平面方向の形状が多角形であると共に最小内径が３μｍ以下の単位構造を規則的に複数配列して形成された前記凹凸構造上の細胞に用いられることを特徴とする（９）記載のスフェロイド形成促進培地。
（１１）　前記細胞外マトリックス成分の含有量が０．０５ｍｇ／ｍｌ以下である（９）又は（１０）いずれか記載のスフェロイド形成促進培地。
（１２）　前記細胞外マトリックス成分の含有量が０．０２ｍｇ／ｍｌ以下である（９）又は（１０）いずれか記載のスフェロイド形成促進培地。
（１３）　前記細胞外マトリックス成分の含有量が０．０１ｍｇ／ｍｌ以下である（９）又は（１０）いずれか記載のスフェロイド形成促進培地。
（１４）　前記細胞外マトリックス成分が基底膜成分であることを特徴とする（９）乃至（１３）いずれか記載のスフェロイド形成促進培地。
（１５）　前記細胞外マトリックス成分が少なくともラミニン、ＩＶ型コラーゲン、ラミニン／エンタクチン複合体から選択される１以上の成分を含むことを特徴とする（９）乃至（１３）いずれか記載のスフェロイド形成促進培地。
（１６）　前記細胞外マトリックス成分がＥｎｇｅｌｂｒｅｔｈ－Ｈｏｌｍ－Ｓｗａｒｍ（ＥＨＳ）マウス腫瘍からの抽出物であることを特徴とする（９）乃至（１３）いずれか記載のスフェロイド形成促進培地。 (1) It is characterized by culturing cells on a culture substrate having a concavo-convex structure that functions as a cell adhesion surface in the presence of a medium basic component and an extracellular matrix component at a concentration that does not gel at the culture temperature. A method for promoting spheroid formation.
(2) The concavo-convex structure is formed by regularly arranging a plurality of unit structures having a width between unit structures of 3 μm or less, a planar shape of a polygon and a minimum inner diameter of 3 μm or less. The method for promoting spheroid formation according to (1).
(3) The method for promoting spheroid formation according to (1) or (2), wherein the content of the extracellular matrix component is 0.05 mg / ml or less.
(4) The method for promoting spheroid formation according to (1) or (2), wherein the content of the extracellular matrix component is 0.02 mg / ml or less.
(5) The method for promoting spheroid formation according to (1) or (2), wherein the content of the extracellular matrix component is 0.01 mg / ml or less.
(6) The method for promoting spheroid formation according to any one of (1) to (5), wherein the extracellular matrix component is a basement membrane component.
(7) The spheroid formation promotion according to any one of (1) to (5), wherein the extracellular matrix component includes at least one component selected from laminin, type IV collagen, and laminin / entactin complex. Method.
(8) The method for promoting spheroid formation according to any one of (1) to (5), wherein the extracellular matrix component is an extract from an Engelbreth-Holm-Swarm (EHS) mouse tumor.
(9) A spheroid that is used for cells on an uneven structure that functions as a cell adhesion surface, and includes a medium basic component and an extracellular matrix component at a concentration that does not gel at the culture temperature. Formation promotion medium.
(10) Used for cells on the concavo-convex structure formed by regularly arranging a plurality of unit structures each having a width between unit structures of 3 μm or less and a planar shape of a polygon and a minimum inner diameter of 3 μm or less. The medium for promoting spheroid formation according to (9), wherein
(11) The spheroid formation promoting medium according to any one of (9) and (10), wherein the content of the extracellular matrix component is 0.05 mg / ml or less.
(12) The spheroid formation promoting medium according to any one of (9) and (10), wherein the content of the extracellular matrix component is 0.02 mg / ml or less.
(13) The spheroid formation promoting medium according to any one of (9) and (10), wherein the content of the extracellular matrix component is 0.01 mg / ml or less.
(14) The spheroid formation promoting medium according to any one of (9) to (13), wherein the extracellular matrix component is a basement membrane component.
(15) The spheroid formation promotion according to any one of (9) to (13), wherein the extracellular matrix component includes at least one component selected from laminin, type IV collagen, and laminin / entactin complex. Culture medium.
(16) The spheroid formation promoting medium according to any one of (9) to (13), wherein the extracellular matrix component is an extract from an Engelbreth-Holm-Swarm (EHS) mouse tumor.
　本発明により、優れたスフェロイドを形成させることができるため、例えば、薬剤のスクリーニングや毒性評価において、単層細胞の混在や分離・崩壊したスフェロイド等に起因するノイズを減らすことができる。また、短期間でスフェロイドを形成させることができるため、コストや手間を省いたスフェロイドの利用が可能となる。 Since excellent spheroids can be formed according to the present invention, for example, in drug screening and toxicity evaluation, it is possible to reduce noise caused by mixing of monolayer cells, separated spheroids, and the like. Moreover, since spheroids can be formed in a short period of time, it is possible to use spheroids that save cost and labor.
細胞接着面の凹凸構造を示す説明図である。It is explanatory drawing which shows the uneven structure of a cell adhesion surface. マトリゲル濃度が０～０．０５ｍｇ／ｍｌの培地で培養したＮＣＩ－Ｈ２０３０細胞の写真である。3 is a photograph of NCI-H2030 cells cultured in a medium with a Matrigel concentration of 0 to 0.05 mg / ml. マトリゲル濃度が０～０．０１ｍｇ／ｍｌの培地で培養したＨｕＨ－７細胞の写真である。2 is a photograph of HuH-7 cells cultured in a medium with a Matrigel concentration of 0 to 0.01 mg / ml. マトリゲル濃度が０～０．０５ｍｇ／ｍｌの培地で培養したＨｓ５７８Ｔ細胞の写真である。2 is a photograph of Hs578T cells cultured in a medium with a Matrigel concentration of 0 to 0.05 mg / ml. マトリゲル濃度が０～０．０５ｍｇ／ｍｌの培地で培養したＡ５４９細胞の写真である。2 is a photograph of A549 cells cultured in a medium having a matrigel concentration of 0 to 0.05 mg / ml. マトリゲル濃度が０～０．０５ｍｇ／ｍｌのＮＣＭ培地およびＨａｍ’ｓ Ｆ－１２Ｋ培地で培養したＰＣ－３細胞の写真である。Fig. 4 is a photograph of PC-3 cells cultured in NCM medium and Ham's F-12K medium having a Matrigel concentration of 0 to 0.05 mg / ml. マトリゲル濃度が０～０．０５ｍｇ／ｍｌのＮＣＭ培地およびＲＰＭＩ培地で培養したＰａｎｃ－１細胞の写真である。2 is a photograph of Panc-1 cells cultured in NCM medium and RPMI medium having a Matrigel concentration of 0 to 0.05 mg / ml. マトリゲル濃度が０～０．０５ｍｇ／ｍｌのＮＣＭ培地およびＲＰＭＩ培地で培養したＢｘＰＣ－３細胞の写真である。2 is a photograph of BxPC-3 cells cultured in NCM medium and RPMI medium having a Matrigel concentration of 0 to 0.05 mg / ml. マトリゲル濃度が０～０．０５ｍｇ／ｍｌのＮＣＭ培地およびＲＰＭＩ培地で培養したｃｏｌｏ２０５細胞の写真である。It is a photograph of colo205 cells cultured in NCM medium and RPMI medium having a Matrigel concentration of 0 to 0.05 mg / ml. マトリゲル濃度が０～０．０５ｍｇ／ｍｌのＮＣＭ培地およびＬ－１５培地で培養したＭＤＡ－ＭＢ－２３１細胞の写真である。2 is a photograph of MDA-MB-231 cells cultured in NCM medium and L-15 medium having a matrigel concentration of 0 to 0.05 mg / ml. マトリゲルの構成成分を添加した培地で培養したＰａｎｃ－１細胞の写真である。It is a photograph of Panc-1 cells cultured in a medium supplemented with Matrigel components. マトリゲルの構成成分を添加した培地で培養したＢｘＰＣ－３細胞の写真である。It is a photograph of BxPC-3 cells cultured in a medium supplemented with Matrigel components. る。マトリゲルの構成成分を添加した培地で培養したｃｏｌｏ２０５細胞の写真である。The It is a photograph of colo205 cells cultured in a medium supplemented with Matrigel components.
　以下、発明を実施するための最良の形態により、本発明を詳説する。 Hereinafter, the present invention will be described in detail according to the best mode for carrying out the invention.
　１．本発明のスフェロイド形成促進方法
　本発明のスフェロイド形成促進方法は、細胞接着面として機能する凹凸構造を有する培養基材上において、培地基礎成分および細胞外マトリックスの存在下で細胞を培養することを特徴とするスフェロイドの形成促進方法である。 1. The Spheroid Formation Promotion Method of the Present Invention The spheroid formation promotion method of the present invention is characterized in that cells are cultured in the presence of a basic medium component and an extracellular matrix on a culture substrate having an uneven structure that functions as a cell adhesion surface. This is a method for promoting the formation of spheroids.
　ここでいうスフェロイドとは、三次元的に細胞同士が集合・凝集化した細胞の集合体を指す。 The spheroid here refers to an aggregate of cells in which cells are aggregated and aggregated three-dimensionally.
　本発明において細胞接着面として機能する凹凸構造の形状は、スフェロイドを形成させる細胞の培養基材への接着性の違いに応じて、線状（ラインアンドスペース）、ピラー状、ホール状等、種々の形状を利用できるが、好ましくは、所定の平面形状からなる単位構造（三角形、四角形、六角形等の多角形、円、その他の形状等）を規則的に複数配列した構造の方が良い。例えば、図１に示すように、平面形状が多角形である単位構造１を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スフェロイドを成長させることができるという点で、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。 The shape of the concavo-convex structure functioning as a cell adhesion surface in the present invention can be various, such as linear (line and space), pillar, hole, etc., depending on the difference in adhesion of the cells that form spheroids to the culture substrate. However, it is preferable to use a structure in which a plurality of unit structures (polygons such as triangles, quadrilaterals, hexagons, circles, and other shapes) having a predetermined planar shape are regularly arranged. For example, as shown in FIG. 1, a plurality of unit structures 1 having a polygonal planar shape can be formed as a continuous structure. At this time, regular polygons such as regular triangles, squares, regular hexagons, and circles are more preferable in that spheroids can be grown on an isotropically uniform structure.
　また、単位構造間の幅（図１、線２の幅）は、３μｍ以下、２μｍ以下、１μｍ以下、７００ｎｍ以下、５００ｎｍ以下、２５０ｎｍ以下というように、小さくなるほど好ましい。この理由としては、単位構造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。 Further, the width between unit structures (the width of FIG. 1, line 2) is preferably as small as 3 μm or less, 2 μm or less, 1 μm or less, 700 nm or less, 500 nm or less, or 250 nm or less. This is because the smaller the width between the unit structures, the more the cells adhered to the concavo-convex structure surface are considered to be able to form spheroids while growing many pseudopods.
　また、単位構造の深さは、スフェロイドを形成させる細胞の培養基材への接着性の違いに応じて、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る。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 depth of the unit structure is 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 depending on the difference in adhesion of the cells for forming spheroids to the culture substrate. It is formed in various sizes as described above. Further, the aspect ratio of the unevenness includes various ones such as 0.2 or more, 0.5 or more, 1 or more, 2 or more.
　また、単位構造の最小内径（好ましくは最大内径）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。ここで、内径とは、単位構造に外接する２本の平行線間の距離を意味し、最小内径とは、単位構造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、単位構造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、単位構造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、単位構造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。 In addition, the minimum inner diameter (preferably the maximum inner diameter) of the unit structure is preferably 3 μm or less, and it is preferable for the same reason as described above to be smaller, such as 2 μm or less, 1 μm or less, 700 nm or less, 500 nm or less, or 250 nm or less. . Here, the inner diameter means the distance between two parallel lines circumscribing the unit structure, and the minimum inner diameter means the shortest distance among the two parallel lines circumscribing the unit structure, The maximum inner diameter means the longest distance between two parallel lines circumscribing the unit structure. For example, when the unit structure is a regular hexagon, the distance between the parallel sides facing each other is the minimum inner diameter, and the distance between the opposite vertices is the maximum inner diameter. When the unit structure is rectangular, the length of the short side is the minimum inner diameter, and the length of the diagonal line is the maximum inner diameter.
　当該構造面を有する市販品としては、例えば、ＮａｎｏＣｕｌｔｕｒｅプレート（登録商標：ＳＣＩＶＡＸ株式会社）が挙げられる。 As a commercially available product having the structural surface, for example, NanoCulture plate (registered trademark: SCIVAX Co., Ltd.) can be mentioned.
　本発明に用いられる培養基材の形状は、細胞を培養できるものであればどのようなものであっても良いが、例えば、フィルム状や基板状（プレート状）に形成でき、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に用いることができる。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 culture substrate used in the present invention may be any shape as long as cells can be cultured. For example, it can be formed into a film shape or a substrate shape (plate shape), such as a petri dish, a dish, It can be used for multiwell plates, flasks, chamber slides and the like. Moreover, the uneven structure should just be formed in at least one part on a base material.
　また、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、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 culture substrate may be any material as long as it is non-toxic to cells. For example, “polystyrene”, “polyethylene”, “polypropylene”, “polyimide”, “polylactic acid or polylactic acid” -Biodegradable polymers such as polyglycolic acid copolymer, polycaprolactone "," cyclic olefin thermoplastic resins such as cyclic olefin copolymer (COC) and cyclic olefin polymer (COP) "," acrylic resin ", “Other resins such as photo-curing resins and thermosetting resins”, “metals such as aluminum oxide”, “glass”, “quartz glass”, “silicon”, etc. can be used, and are made of silicon, glass, etc. A substrate in which a coating layer such as “resin”, “photoresist”, or “metal such as aluminum oxide” is formed on the surface of the substrate body can also be used.
　さらに、培養基材の表面は、親水性の制御がなされているものであってもよい。制御方法としては、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、二酸化ケイ素（ＳｉＯ２）、ポリリジン、各種細胞外マトリックスによる被覆等が挙げられるが、これらに限定されるものではない。 Furthermore, the surface of the culture substrate may be one whose hydrophilicity is controlled. Examples of the control method include, but are not limited to, surface modification technology by irradiation with ultraviolet rays, electron beams, gamma rays, plasma, etc., coating with silicon dioxide (SiO 2 ), polylysine, various extracellular matrices, and the like. It is not a thing.
　なお、細胞培養基材の製造方法は、凹凸構造を形成し得る方法であればいかなる方法でもよく、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。 The cell culture substrate can be produced by any method as long as it can form a concavo-convex structure. For example, nanoimprint technology, solution casting method, etching, blasting, corona discharge and 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.
　本発明において培地基礎成分とは、細胞が同化し得る炭素源、消化しうる窒素源および無機塩から構成されるものであり、具体的には例えば無機塩類、アミノ酸、糖質、およびビタミン類を含むものである。 In the present invention, the medium basic component is composed of a carbon source that can be assimilated by cells, a nitrogen source that can be digested, and an inorganic salt, and specifically includes, for example, inorganic salts, amino acids, carbohydrates, and vitamins. Is included.
　ミネラル類としては、例えば、カルシウム、カリウム、マグネシウム、リン、ナトリウム、銅、鉄、セレン、マンガン、ケイ素、モリブデン、バナジウム、ニッケルおよび亜鉛等が挙げられるが、これらに限定されるものではない。 Examples of minerals include, but are not limited to, calcium, potassium, magnesium, phosphorus, sodium, copper, iron, selenium, manganese, silicon, molybdenum, vanadium, nickel, and zinc.
　アミノ酸としては、例えば、Ｌ－アラニン、Ｌ－アルギニン、Ｌ－アスパラギン、Ｌ－アスパラギン酸、Ｌ－システイン、Ｌ－グルタミン酸、Ｌ－グルタミン、グリシン、Ｌ－ヒスチジン、Ｌ－イソロイシン、Ｌ－ロイシン、Ｌ－リジン、Ｌ－メチオニン、Ｌ－フェニルアラニン、Ｌ－プロリン、Ｌ－セリン、Ｌ－スレオニン、Ｌ－トリプトファン、Ｌ－チロシン、Ｌ－バリン等が挙げられるが、これらに限定されるものではない。 Examples of amino acids include L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine, L-glutamic acid, L-glutamine, glycine, L-histidine, L-isoleucine, L-leucine, L Examples include, but are not limited to, lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, and L-valine.
　糖質としては、例えば、グルコース、ガラクトース、マンノース、フルクトース等が挙げられるが、これらに限定されるものではない。 Examples of carbohydrates include, but are not limited to, glucose, galactose, mannose, fructose and the like.
　ビタミン類としては、例えば、Ｄ－ビオチン、フォリニック酸、ＤＬ－α－リポ酸、ニコチンアミド、Ｄ－パントテン酸、ピリドキシン、リボフラビンン、チアミン、シアノコバラミン（ビタミンＢ１２）等が挙げられるが、これらに限定されるものではない。 Examples of vitamins include D-biotin, folinic acid, DL-α-lipoic acid, nicotinamide, D-pantothenic acid, pyridoxine, riboflavin, thiamine, cyanocobalamin (vitamin B 12 ), etc. It is not limited to.
　培地基礎成分を含む基礎培地としては、例えば、イーグル培地（例えば、ＢＭＥ、ＭＥＭ，ＤＭＥＭ、ＧＭＥＭ）、ＲＰＭＩ培地、Ｌ－１５培地、フィッシャー培地、ハム培地（例えば、Ｆ１０、Ｆ１２）、ＭＣＤＢ培地等が挙げられるが、これらに限定されるものではない。 Examples of the basal medium containing the basal medium include eagle medium (for example, BME, MEM, DMEM, GMEM), RPMI medium, L-15 medium, Fisher medium, ham medium (for example, F10, F12), MCDB medium, and the like. However, it is not limited to these.
　本発明において細胞外マトリックス成分とは、生体内において細胞を取り巻いている物質であり、骨格的役割、細胞接着における足場の役割、細胞増殖因子などを保持・提供する役割等を担う物質またはそれらと似た生物活性を示す物質を指す。 In the present invention, the extracellular matrix component is a substance that surrounds cells in the living body, and is a substance that plays a skeletal role, a role of a scaffold in cell adhesion, a role of holding / providing a cell growth factor, or the like. It refers to a substance that exhibits similar biological activity.
　本発明のスフェロイド形成促進方法に用いる細胞外マトリックス成分としては、例えば、コラーゲン、エラスチン、フィブリリンなどの繊維性蛋白質、ヒアルロン酸、コンドロイチン硫酸、デルマタン硫酸、へパラン硫酸、ヘパリン、ケタラン硫酸などのグリコサミノグリカンとプロテオグリカン、フィブロネクチン、ラミニン、ビトロネクチン、テネイシン、トロンボスポンジン、エンタクチン、ニドゲン、オステオポンチン、フォンビルブラント因子、フィブリノーゲンなどの細胞接着性蛋白質が挙げられるが、生体内において細胞を取り巻く物質であれば特に限定されるものではない。また、細胞外マトリックスは人工的に作られたものであってもよい。本発明では、これらの中の単独を用いても、複数を併用して用いてもよい。 Examples of extracellular matrix components used in the method for promoting spheroid formation of the present invention include fibrous proteins such as collagen, elastin, and fibrillin, and glycosami such as hyaluronic acid, chondroitin sulfate, dermatan sulfate, heparan sulfate, heparin, and ketalan sulfate. Cell adhesion proteins such as noglycan and proteoglycan, fibronectin, laminin, vitronectin, tenascin, thrombospondin, entactin, nidogen, osteopontin, von Willebrand factor, fibrinogen, etc. It is not particularly limited. The extracellular matrix may be artificially made. In the present invention, these may be used alone or in combination.
　また、本発明のスフェロイド形成促進方法に用いる細胞外マトリックス成分としては、基底膜成分を用いることができる。基底膜は、細胞の接着、運動性、増殖、機能維持、分化を含む様々なプロセスにおいて重要な役割を果たすことが知られている。代表的な基底膜成分としては、ラミニン、ＩＶ型コラーゲン、エンタクチン、ヘパリン、へパラン硫酸プロテオグリカンが挙げられる。 In addition, as an extracellular matrix component used in the spheroid formation promoting method of the present invention, a basement membrane component can be used. The basement membrane is known to play an important role in various processes including cell adhesion, motility, proliferation, functional maintenance, and differentiation. Typical basement membrane components include laminin, type IV collagen, entactin, heparin, and heparan sulfate proteoglycan.
　さらに、本発明のスフェロイド形成促進方法に用いる細胞外マトリックス成分としては、動物からの抽出物またはその調製物を用いることができ、例えば、Ｅｎｇｅｌｂｒｅｔｈ－Ｈｏｌｍ－Ｓｗａｒｍ（ＥＨＳ）マウス腫瘍からの抽出物を用いることができる。当該抽出物から調製された市販品の例としては、「マトリゲル」（登録商標：ＢＤ Ｂｉｏｓｃｉｅｎｃｅ社）やＥＣＭゲル（Ｓｉｇｍａ－Ａｌｄｒｉｃｈ社）が挙げられる。このとき、調製物中に含まれる成分の組成および含有量については特に限定されないが、シグナル研究、増殖因子の機能解明、遺伝子発現研究、薬剤スクリーニング等の研究目的で使用される場合には、各種増殖因子の含有量が低減されていることが望ましい。 Furthermore, as an extracellular matrix component used in the method for promoting spheroid formation of the present invention, an extract from an animal or a preparation thereof can be used. For example, an extract from an Engelbreth-Holm-Swarm (EHS) mouse tumor is used. Can be used. Examples of commercially available products prepared from the extract include “Matrigel” (registered trademark: BD Bioscience) and ECM gel (Sigma-Aldrich). At this time, the composition and content of the components contained in the preparation are not particularly limited, but when used for research purposes such as signal research, growth factor function elucidation, gene expression research, drug screening, etc. It is desirable that the growth factor content is reduced.
　さらに、本発明のスフェロイド形成促進方法に用いる細胞外マトリックス成分としては、上記「マトリゲル」の構成成分であるラミニン、ＩＶ型コラーゲン、ラミニン／エンタクチン複合体を単独で又は複数を併用して用いることができる。精製された各構成成分を用いることにより、各成分の添加量を容易にコントロールすることができる。また、ロット差が大きいために安定した結果が得られないという「マトリゲル」のデメリットを解消することができる。 Furthermore, as the extracellular matrix component used in the method for promoting spheroid formation of the present invention, laminin, type IV collagen, laminin / entactin complex, which is a constituent component of the “Matrigel”, may be used alone or in combination. it can. By using each purified component, the amount of each component added can be easily controlled. Further, the disadvantage of “Matrigel” that a stable result cannot be obtained due to a large lot difference can be solved.
　なお、本発明のスフェロイド形成促進方法において、細胞外マトリックス成分の培地中の含有量は、スフェロイド形成時の培養温度においてゲル化しない濃度であれば、培養する細胞の種類、基礎培地成分の種類等に応じて適宜変更可能である。ゲル化しない濃度とは、培地交換や各種アッセイ、細胞回収等のハンドリングに影響を及ぼさない濃度、かつ、培養後にスムースに評価系に移行できる濃度のことを指す。具体的には、例えば１０％以下である。 In the method for promoting spheroid formation according to the present invention, if the content of the extracellular matrix component in the medium is a concentration that does not gel at the culture temperature during spheroid formation, the type of cells to be cultured, the type of basal medium component, etc. It can be appropriately changed depending on the situation. The concentration that does not cause gelation refers to a concentration that does not affect handling such as medium exchange, various assays, cell recovery, and the like, and a concentration that can be smoothly transferred to an evaluation system after culture. Specifically, it is 10% or less, for example.
　スフェロイド形成促進に有効な細胞外マトリックス成分の培地中の含有量は、例えば、培地量の０．０５ｍｇ／ｍｌ以下であり、好ましくは０．０２ｍｇ／ｍｌ以下、０．０１ｍｇ／ｍｌ以下、０．００５ｍｇ／ｍｌ以下である。 The content of the extracellular matrix component effective for promoting spheroid formation in the medium is, for example, 0.05 mg / ml or less, preferably 0.02 mg / ml or less, 0.01 mg / ml or less, 0. 005 mg / ml or less.
　本発明のスフェロイド形成促進方法は、基礎培地成分および細胞外マトリックス成分の他に、さらにウシ、ウマ、ヒト等の血清や、各種増殖因子（例えば、ＥＧＦ、ｂＦＧＦ、ＮＧＦ、ＰＤＧＦ、ＩＧＦ－１、ＴＧＦ－β、ＶＥＧＦ、ＴＮＦ－α）、細胞の増殖および維持に有効であることが知られている物質（例えば、血清アルブミン、トランスフェリン、脂質、脂質酸源、コレステロール、ピルビン酸、グルココルチコイド、ＤＮＡおよびＲＮＡ合成用ヌクレオシド）、微量金属元素、界面活性剤、サイトカイン、分化誘導因子等を用いてもよい。これら添加物は、培養する細胞の種類に応じて適宜選択して添加することができる。さらに、必要に応じて、ストレプトマイシン、ペニシリンＧカリウムおよびゲンタマイシン等の抗生物質や、フェノールレッド等のｐＨ指示薬を添加しても良い。 In addition to the basal medium component and extracellular matrix component, the spheroid formation promotion method of the present invention further includes serum from bovine, horse, human, etc., and various growth factors (for example, EGF, bFGF, NGF, PDGF, IGF-1, TGF-β, VEGF, TNF-α), substances known to be effective in cell growth and maintenance (eg, serum albumin, transferrin, lipid, lipid acid source, cholesterol, pyruvate, glucocorticoid, DNA And RNA synthesis nucleosides), trace metal elements, surfactants, cytokines, differentiation-inducing factors, and the like. These additives can be appropriately selected and added according to the type of cells to be cultured. Furthermore, antibiotics such as streptomycin, penicillin G potassium and gentamicin, and pH indicators such as phenol red may be added as necessary.
　ところで、凹凸構造面上における培養では、細胞種によりスフェロイドの形成率が異なる。これは細胞が有する培養基材への接着性の違いに起因するものと考えられる。そのため、細胞の培養基材への接着を制御するために、細胞種に応じて、凹凸構造の平面形状、単位構造間の幅、培養基材の材質、親水性等の調節や用いる培地の基礎培地成分の調節を行い、スフェロイドの形成率を向上させることが可能である。 By the way, in the culture on the concavo-convex structure surface, the spheroid formation rate varies depending on the cell type. This is considered to be due to the difference in the adhesion of the cells to the culture substrate. Therefore, in order to control the adhesion of cells to the culture substrate, depending on the cell type, the planar shape of the concavo-convex structure, the width between unit structures, the material of the culture substrate, the hydrophilicity, etc. and the basis of the medium used It is possible to improve the rate of spheroid formation by adjusting the medium components.
　一方、本発明のスフェロイド形成促進方法においては、培地に含まれる所定量の細胞外マトリックス成分が、細胞に物理的な支持をもたらし、また、細胞表面の受容体等を介して生化学的な刺激をもたらすことにより、細胞間接着を増強するものと思われる。そのため、細胞間の接着力と細胞と培養基材間の接着力との間のバランスに変化が生じ、スフェロイドの形成が促進されるものと考えられる。 On the other hand, in the spheroid formation promotion method of the present invention, a predetermined amount of extracellular matrix components contained in the medium provide physical support to the cells, and biochemical stimulation via receptors on the cell surface and the like. It seems to enhance cell-cell adhesion. For this reason, it is considered that a change occurs in the balance between the adhesion between cells and the adhesion between the cells and the culture substrate, and the formation of spheroids is promoted.
　このことから、細胞種に応じて、凹凸構造の平面形状、単位構造間の幅、培養基材の材質、親水性等の調節、培地基礎成分の調節、培地に含まれる細胞外マトリックスの成分や含有量の調節を適宜組み合わせることにより、ノイズの少ない優れたスフェロイド群の形成が可能となる。 From this, depending on the cell type, the planar shape of the concavo-convex structure, the width between unit structures, the material of the culture substrate, the adjustment of the hydrophilicity, the adjustment of the basic components of the medium, the components of the extracellular matrix contained in the medium, By appropriately combining the adjustment of the content, an excellent spheroid group with less noise can be formed.
　本発明において用いられる細胞は、スフェロイドを形成できる細胞であればどのようなものであってもよいが、動物由来の細胞であることが好ましく、その中でも哺乳類由来の細胞であることが好ましい。哺乳類動物としては、例えば、ヒト、イヌ、ネコ、サル、ウシ、ブタ、ヒツジ、ウマ、ラット、マウス等が挙げられるが、これらに限定されるものではない。 The cell used in the present invention may be any cell as long as it can form a spheroid, but is preferably an animal-derived cell, and more preferably a mammal-derived cell. Examples of mammals include, but are not limited to, humans, dogs, cats, monkeys, cows, pigs, sheep, horses, rats, mice and the like.
　本発明において用いられる細胞は、造血系細胞や間葉系細胞を含む中胚葉組織細胞、内胚葉組織細胞、外胚葉組織細胞あるいは受精卵からこれらの細胞へ分化する過程に含まれるあらゆる細胞、および胚性幹細胞などの幹細胞のいずれでもよい。造血系細胞とは、例えば、造血幹細胞、造血前駆細胞、赤血球細胞、リンパ球細胞、顆粒球細胞、血小板細胞などを指す。間葉系細胞とは、骨細胞、軟骨細胞、筋細胞、腱細胞、脂肪細胞、毛乳頭細胞、歯髄細胞などの組織学的にいうところの結合組織の細胞およびこれらの細胞に分化する能力を有する細胞を指す。かかる細胞は、培養細胞として株化されたものであっても、生物組織から得られた初代細胞であってもよい。また、例えば、腫瘍化した細胞、遺伝子工学的手法により形質転換された細胞やウイルスベクターにより感染された細胞等であってもよい。具体的には、上皮系細胞、例えば初代肝細胞、肝細胞株細胞、初代癌細胞、癌細胞株細胞が好ましく、例えば、Ａ５４９細胞（ヒト肺癌細胞株）、ＨｕＨ－７細胞（ヒト肝癌細胞株）、ＰＣ－３細胞（ヒト前立腺癌細胞株）、ＮＣＩ－Ｈ２０３０細胞（ヒト肺腺癌細胞株）、Ｈｓ５７８Ｔ細胞（ヒト乳癌細胞株）、Ｐａｎｃ－１細胞（ヒト膵癌細胞株）、ＢｘＰＣ－３細胞（ヒト膵癌細胞株）、ｃｏｌｏ２０５細胞（ヒト大腸癌細胞株）、ＭＤＡ－ＭＢ－２３１細胞（ヒト乳癌細胞株）等が好適な細胞として挙げられる。 The cells used in the present invention include mesoderm tissue cells including hematopoietic cells and mesenchymal cells, endoderm tissue cells, ectoderm tissue cells or any cells included in the process of differentiation from fertilized eggs to these cells, and Any of stem cells such as embryonic stem cells may be used. Examples of hematopoietic cells include hematopoietic stem cells, hematopoietic progenitor cells, red blood cells, lymphocyte cells, granulocyte cells, and platelet cells. Mesenchymal cells are cells of connective tissues such as bone cells, chondrocytes, muscle cells, tendon cells, adipocytes, dermal papilla cells, pulp cells, and the ability to differentiate into these cells. It refers to the cell that has it. Such cells may be established as cultured cells or primary cells obtained from biological tissues. Further, for example, it may be a tumor cell, a cell transformed by a genetic engineering technique, a cell infected by a viral vector, or the like. Specifically, epithelial cells such as primary hepatocytes, hepatocyte cell lines, primary cancer cells and cancer cell lines are preferred, such as A549 cells (human lung cancer cell lines), HuH-7 cells (human hepatoma cell lines). ), PC-3 cells (human prostate cancer cell line), NCI-H2030 cells (human lung adenocarcinoma cell line), Hs578T cells (human breast cancer cell line), Panc-1 cells (human pancreatic cancer cell line), BxPC-3 Suitable cells include cells (human pancreatic cancer cell line), colo205 cells (human colon cancer cell line), MDA-MB-231 cells (human breast cancer cell line) and the like.
　本発明において、培地基礎成分と細胞外マトリックス成分とを共存させる方法は、特に限定されず、培地基礎成分に対して細胞外マトリックス成分を添加してもよく、また、容器に対して培地基礎成分と細胞外マトリックス成分を同時に共存させてもよい。例えば、培地基礎成分および所定の細胞外マトリックス成分を含む培地を利用することができる。 In the present invention, the method for allowing the medium basic component and the extracellular matrix component to coexist is not particularly limited, and the extracellular matrix component may be added to the medium basic component, and the medium basic component may be added to the container. And extracellular matrix components may coexist simultaneously. For example, a medium containing a medium basic component and a predetermined extracellular matrix component can be used.
　また、培地基礎成分および／または細胞外マトリックス成分と細胞とを共存させる方法は、例えば、培地基礎成分および細胞外マトリックス成分を含む培地に細胞を添加する方法、基礎培地成分を含む培地内における細胞培養の途中で細胞外マトリックス成分を添加する方法等が挙げられる。 The medium basic component and / or the extracellular matrix component and the cell can coexist with, for example, a method of adding cells to a medium containing the medium basic component and extracellular matrix component, or a cell in the medium containing the basic medium component. Examples thereof include a method of adding an extracellular matrix component during the culture.
　このとき、培地基礎成分および細胞外マトリックス成分と細胞とを共存させるタイミングは特に限定されるものではなく、例えば、培養開始から終了まで全ての期間でも良く、一部の期間であってもよいが、スフェロイドの形成能を維持させるためには、前者の期間で用いる方が好都合である。 At this time, the timing at which the medium basic component and extracellular matrix component coexist with the cells is not particularly limited. For example, the entire period from the start to the end of the culture or a part of the period may be used. In order to maintain the ability to form spheroids, it is more convenient to use the former period.
　培養条件は、常法に従えば良く、特に制限されるものではない。例えば、培地中の酸素濃度、浸透圧、ｐＨ、培地温度等は、培養する細胞の種類、培養目的、培養量、基礎培地成分の種類等に応じて適宜変更することができる。例えば、気相のＣＯ２濃度が０－４０％の雰囲気下、３７℃程度で、３－１４日間培養すればよい。 Culture conditions are not particularly limited as long as they follow conventional methods. For example, the oxygen concentration, osmotic pressure, pH, medium temperature, and the like in the medium can be appropriately changed according to the type of cells to be cultured, the purpose of culture, the culture amount, the type of basal medium components, and the like. For example, it may be cultured for 3-14 days at about 37 ° C. in an atmosphere where the gas phase CO 2 concentration is 0-40%.
　２．本発明のスフェロイド形成促進培地
　本発明のスフェロイド形成促進培地は、細胞接着面として機能する凹凸構造を有する培養基材に用いられるものであって、培地基礎成分と、所定の細胞外マトリックスとを含有することを特徴とするスフェロイドの形成促進培地である。 2. Spheroid formation promoting medium of the present invention The spheroid formation promoting medium of the present invention is used for a culture substrate having a concavo-convex structure that functions as a cell adhesion surface, and contains a medium basic component and a predetermined extracellular matrix. A medium for promoting the formation of spheroids.
　本発明のスフェロイド形成促進培地における凹凸構造の形状および培養基材とその製造方法については、前述と同様の物および方法を用いることができる。 For the shape of the concavo-convex structure in the spheroid formation promoting medium of the present invention, the culture substrate and the production method thereof, the same products and methods as described above can be used.
　本発明のスフェロイド形成促進培地における培地基礎成分、細胞外マトリックス成分、および培地中の細胞外マトリックス成分の含有量については、前述と同様の物を用いることができる。 As the medium basic component, extracellular matrix component, and extracellular matrix component content in the medium in the spheroid formation promoting medium of the present invention, the same materials as described above can be used.
　以下、実施例により本発明を具体的に説明する。ただし、本発明は以下の記述に限定されるものではない。 Hereinafter, the present invention will be specifically described by way of examples. However, the present invention is not limited to the following description.
　マトリゲル（登録商標　ＢＤ Ｂｉｏｓｃｉｅｎｃｅ社製ＢＤマトリゲルグロースファクターリデュースト）を０～５．０％濃度となるよう培地（ＳＣＩＶＡＸ社製 ＮＣＭ－Ｍ）に添加し、当該培地中でＮＣＩ－Ｈ２０３０細胞（ＣＲＬ－５９１４：ヒト肺腺癌細胞株）
を７日間培養した。培養は、ＮＣＩ－Ｈ２０３０細胞を１×１０４ｃｅｌｌｓ／１００μｌ／ｗｅｌｌで９６ｗｅｌｌプレートに播種し、３７℃、５％ ＣＯ２条件下にて行った。９６ｗｅｌｌプレートとしては、細胞接着面上に凹凸構造を有したプレート（ＳＣＩＶＡＸ社製 ＮａｎｏＣｕｌｔｕｒｅプレート：凹凸構造面の材質＝環状オレフィン重合体（ＣＯＰ）、凹凸構造の平面形状＝正六角形、単位構造間の幅（線幅）＝７００ｎｍ、単位構造の最小内径＝３μｍ、深さ＝１μｍ）を用いた。 Matrigel (registered trademark, BD Bioscience, BD Matrigel Growth Factor Reduce) was added to a medium (SCVAX NCM-M) to a concentration of 0 to 5.0%, and NCI-H2030 cells (CRL-) were added in the medium. 5914: Human lung adenocarcinoma cell line)
Was cultured for 7 days. Culturing was performed by seeding NCI-H2030 cells at 1 × 10 4 cells / 100 μl / well in a 96-well plate under conditions of 37 ° C. and 5% CO 2 . The 96-well plate is a plate having a concavo-convex structure on the cell adhesion surface (NanoCulture plate manufactured by SCIVAX: material of the concavo-convex structure surface = cyclic olefin polymer (COP), planar shape of the concavo-convex structure = regular hexagon, between unit structures The width (line width) = 700 nm, the minimum inner diameter of the unit structure = 3 μm, and the depth = 1 μm were used.
　　培養７日目に、各マトリゲル濃度におけるスフェロイドの形成能を光学顕微鏡にて観察した。光学顕微鏡写真を図２に示す。 On the seventh day of culture, the ability to form spheroids at each Matrigel concentration was observed with an optical microscope. An optical micrograph is shown in FIG.
　マトリゲルを添加していない培地（マトリゲル濃度０ｖｏｌ％）ではほとんどスフェロイドが形成されなかった。これに対し、マトリゲルを０．１～１．０ｖｏｌ％添加した培地では小さなスフェロイドが形成され、濃度依存的にその数が向上していた。さらに、マトリゲル濃度２．０％～５．０ｖｏｌ％では大きなスフェロイドが形成された。 Spheroids were hardly formed in the medium to which no Matrigel was added (Matrigel concentration 0 vol%). On the other hand, small spheroids were formed in the medium to which Matrigel was added in an amount of 0.1 to 1.0 vol%, and the number thereof increased depending on the concentration. Furthermore, large spheroids were formed at a Matrigel concentration of 2.0% to 5.0 vol%.
　マトリゲル（登録商標　ＢＤ Ｂｉｏｓｃｉｅｎｃｅ社製ＢＤマトリゲルグロースファクターリデュースト）を０～１．０ｖｏｌ％濃度となるよう培地（ＳＣＩＶＡＸ社製 ＮＣＭ－Ｍ）に添加し、当該培地中でＨｕＨ－７細胞（ＲＣＢ１３６６：ヒト肝癌由来の培養細胞株）を７日間培養した。培養は、ＨｕＨ－７細胞を１×１０４ｃｅｌｌｓ／１００μｌ／ｗｅｌｌで９６ｗｅｌｌプレートに播種し、３７℃、５％ ＣＯ２条件下にて行った。９６ｗｅｌｌプレートとしては、細胞接着面上に凹凸構造を有したプレート（ＳＣＩＶＡＸ社製 ＮａｎｏＣｕｌｔｕｒｅプレート：凹凸構造面の材質＝環状オレフィン重合体（ＣＯＰ）、凹凸構造の平面形状＝正四角形、単位構造間の幅（線幅）＝７００ｎｍ、単位構造の最小内径＝３μｍ、深さ＝１μｍ）を用いた。 Matrigel (registered trademark BD Bioscience BD Matrigel Growth Factor Reduced) was added to a medium (SCIVAX NCM-M) to a concentration of 0 to 1.0 vol%, and HuH-7 cells (RCB1366: A cultured cell line derived from human liver cancer) was cultured for 7 days. The culture was performed by seeding HuH-7 cells in 96-well plates at 1 × 10 4 cells / 100 μl / well at 37 ° C. under 5% CO 2 conditions. The 96-well plate is a plate having a concavo-convex structure on the cell adhesion surface (NanoCulture plate manufactured by SCIVAX: material of the concavo-convex structure surface = cyclic olefin polymer (COP), planar shape of the concavo-convex structure = rectangular, between unit structures The width (line width) = 700 nm, the minimum inner diameter of the unit structure = 3 μm, and the depth = 1 μm were used.
　培養７日目に、各マトリゲル濃度におけるスフェロイドの形成能を光学顕微鏡にて観察した。光学顕微鏡写真を図３に示す。 On the seventh day of culture, the ability to form spheroids at each Matrigel concentration was observed with an optical microscope. An optical micrograph is shown in FIG.
　マトリゲルを添加していない培地（マトリゲル濃度０ｖｏｌ％）では、ほとんどスフェロイドを形成されず、マトリゲル濃度０．２％～０．５ｖｏｌ％ではスフェロイドを形成する細胞の他に二次元に成長・増殖する細胞が混在していた。これに対し、マトリゲル濃度１．０ｖｏｌ％では二次元に成長・増殖する細胞が減少し、スフェロイドの数・大きさ共に向上した。 Spheroids are hardly formed in the medium to which Matrigel is not added (Matrigel concentration 0 vol%), and cells that grow and proliferate in two dimensions in addition to cells that form spheroids at a matrigel concentration of 0.2% to 0.5 vol%. Were mixed. In contrast, when the Matrigel concentration was 1.0 vol%, the number of cells that grew and proliferated in two dimensions decreased, and both the number and size of spheroids were improved.
　マトリゲル（登録商標　ＢＤ Ｂｉｏｓｃｉｅｎｃｅ社製ＢＤマトリゲルグロースファクターリデュースト）を０～５．０ｖｏｌ％濃度となるよう培地（ＳＣＩＶＡＸ社製 ＮＣＭ－Ｍ）に添加し、当該培地中でＨｓ５７８Ｔ細胞（ＨＴＢ－１２６：ヒト乳癌細胞株）
を７日間培養した。培養は、Ｈｓ５７８Ｔ細胞を１×１０４ｃｅｌｌｓ／１００μｌ／ｗｅｌｌで９６ｗｅｌｌプレートに播種し、３７℃、５％ ＣＯ２条件下にて行った。９６ｗｅｌｌプレートとしては、細胞接着面上に凹凸構造を有したプレート（ＳＣＩＶＡＸ社製 ＮａｎｏＣｕｌｔｕｒｅプレート：凹凸構造面の材質＝環状オレフィン重合体（ＣＯＰ）、凹凸構造の平面形状＝正六角形、単位構造間の幅（線幅）＝７００ｎｍ、単位構造の最小内径＝３μｍ、深さ＝１μｍ）を用いた。 Matrigel (registered trademark BD Bioscience BD Matrigel Growth Factor Reduce) was added to a medium (SCIVAX NCM-M) to a concentration of 0 to 5.0 vol%, and Hs578T cells (HTB-126: Human breast cancer cell line)
Was cultured for 7 days. Culturing was performed by seeding Hs578T cells at 1 × 10 4 cells / 100 μl / well in a 96-well plate at 37 ° C. and 5% CO 2 . The 96-well plate is a plate having a concavo-convex structure on the cell adhesion surface (NanoCulture plate manufactured by SCIVAX: material of the concavo-convex structure surface = cyclic olefin polymer (COP), planar shape of the concavo-convex structure = regular hexagon, between unit structures The width (line width) = 700 nm, the minimum inner diameter of the unit structure = 3 μm, and the depth = 1 μm were used.
　　培養７日目に、各マトリゲル濃度におけるスフェロイドの形成能を光学顕微鏡にて観察した。光学顕微鏡写真を図４に示す。 On the seventh day of culture, the ability to form spheroids at each Matrigel concentration was observed with an optical microscope. An optical micrograph is shown in FIG.
　マトリゲルを添加しない培地（マトリゲル濃度０ｖｏｌ％）においてもスフェロイドは形成されたが、マトリゲル濃度１．０ｖｏｌ％以上、特にマトリゲル濃度１．０％～２．０ｖｏｌ％では、より大きなスフェロイドが形成された。 Spheroids were formed even in a medium without addition of Matrigel (Matrigel concentration 0 vol%), but larger spheroids were formed at a Matrigel concentration of 1.0 vol% or more, particularly at a Matrigel concentration of 1.0% to 2.0 vol%.
　マトリゲル（登録商標　ＢＤ Ｂｉｏｓｃｉｅｎｃｅ社製ＢＤマトリゲルグロースファクターリデュースト）を０～５．０ｖｏｌ％濃度となるよう培地（ＳＣＩＶＡＸ社製 ＮＣＭ－Ｍ）に添加し、当該培地中でＡ５４９細胞（ＲＣＢ００９８：ヒト肺癌由来の培養細胞株）を７日間培養した。培養は、Ａ５４９細胞を１×１０４ｃｅｌｌｓ／１００μｌ／ｗｅｌｌで９６ｗｅｌｌプレートに播種し、３７℃、５％ ＣＯ２条件下にて行った。９６ｗｅｌｌプレートとしては、細胞接着面上に凹凸構造を有したプレート（ＳＣＩＶＡＸ社製 ＮａｎｏＣｕｌｔｕｒｅプレート：凹凸構造面の材質＝環状オレフィン重合体（ＣＯＰ）、凹凸構造の平面形状＝正四角形、単位構造間の幅（線幅）＝７００ｎｍ、単位構造の最小内径＝３μｍ、深さ＝１μｍ）を用いた。 Matrigel (registered trademark BD Bioscience BD Matrigel Growth Factor Reduced) was added to a medium (SCIVAX NCM-M) to a concentration of 0 to 5.0 vol%, and A549 cells (RCB0098: human lung cancer) were added to the medium. The cultured cell line was cultured for 7 days. Cultivation was performed by seeding A549 cells at 1 × 10 4 cells / 100 μl / well in a 96-well plate under conditions of 37 ° C. and 5% CO 2 . The 96-well plate is a plate having a concavo-convex structure on the cell adhesion surface (NanoCulture plate manufactured by SCIVAX: material of the concavo-convex structure surface = cyclic olefin polymer (COP), planar shape of the concavo-convex structure = rectangular, between unit structures The width (line width) = 700 nm, the minimum inner diameter of the unit structure = 3 μm, and the depth = 1 μm were used.
　培養７日目に、各マトリゲル濃度におけるスフェロイドの形成能を光学顕微鏡にて観察した。光学顕微鏡写真を図５に示す。 On the seventh day of culture, the ability to form spheroids at each Matrigel concentration was observed with an optical microscope. An optical micrograph is shown in FIG.
　マトリゲルを添加しない培地（マトリゲル濃度０ｖｏｌ％）と比較し、マトリゲルを添加した培地では良好なスフェロイドが形成された。特にマトリゲル濃度１．０％～５．０ｖｏｌ％では大きなスフェロイドが形成された。 Favorable spheroids were formed in the medium to which Matrigel was added, compared to the medium to which Matrigel was not added (Matrigel concentration 0 vol%). In particular, large spheroids were formed at a matrigel concentration of 1.0% to 5.0 vol%.
　マトリゲル（登録商標　ＢＤ Ｂｉｏｓｃｉｅｎｃｅ社製ＢＤマトリゲルグロースファクターリデュースト）を０～５．０ｖｏｌ％濃度となるよう培地（ＳＣＩＶＡＸ社製 ＮＣＭ－ＭまたはＷａｋｏ社製
 Ｈａｍ’ｓ Ｆ１２－Ｋ）に添加し、当該培地中でＰＣ－３細胞（ＣＲＬ１４３５：ヒト前立腺癌由来の培養細胞株）を７日間培養した。培養は、ＰＣ－３細胞を１×１０４ｃｅｌｌｓ／１００μｌ／ｗｅｌｌで９６ｗｅｌｌプレートに播種し、３７℃、５％ ＣＯ２条件下にて行った。９６ｗｅｌｌプレートとしては、細胞接着面上に凹凸構造を有したプレート（ＳＣＩＶＡＸ社製 ＮａｎｏＣｕｌｔｕｒｅプレート：凹凸構造面の材質＝環状オレフィン重合体（ＣＯＰ）、凹凸構造の平面形状＝正四角形、単位構造間の幅（線幅）＝７００ｎｍ、単位構造の最小内径＝３μｍ、深さ＝１μｍ）を用いた。 Matrigel (registered trademark BD Bioscience BD Matrigel Growth Factor Reduced) is added to a medium (SCIVAX NCM-M or Wako Ham's F12-K) to a concentration of 0 to 5.0 vol%, and PC-3 cells (CRL1435: cultured cell line derived from human prostate cancer) were cultured in the medium for 7 days. Cultivation was performed by seeding PC-3 cells in 96-well plates at 1 × 10 4 cells / 100 μl / well at 37 ° C. under 5% CO 2 conditions. The 96-well plate is a plate having a concavo-convex structure on the cell adhesion surface (NanoCulture plate manufactured by SCIVAX: material of the concavo-convex structure surface = cyclic olefin polymer (COP), planar shape of the concavo-convex structure = rectangular, between unit structures The width (line width) = 700 nm, the minimum inner diameter of the unit structure = 3 μm, and the depth = 1 μm were used.
　培養７日目に、各マトリゲル濃度におけるスフェロイドの形成能を光学顕微鏡にて観察した。光学顕微鏡写真を図６に示す。 On the seventh day of culture, the ability to form spheroids at each Matrigel concentration was observed with an optical microscope. An optical micrograph is shown in FIG.
　マトリゲルを添加していない培地（マトリゲル濃度０ｖｏｌ％）では、ほとんどスフェロイドが形成されなかった。これに対し、マトリゲルを添加したＮＣＭ培地では、マトリゲル濃度１．０％～５．０ｖｏｌ％でスフェロイドが形成され、特にマトリゲル１．０ｖｏｌ％の時に最も良好なスフェロイドが形成された。また、マトリゲルを添加したＨａｍ’ｓ Ｆ１２－Ｋ培地においてもスフェロイド様の細胞塊が形成された。 Spheroids were hardly formed in the medium to which no Matrigel was added (Matrigel concentration 0 vol%). On the other hand, in the NCM medium to which Matrigel was added, spheroids were formed at a Matrigel concentration of 1.0% to 5.0 vol%, and the best spheroids were formed particularly when Matrigel was 1.0 vol%. In addition, spheroid-like cell clusters were also formed in Ham's F12-K medium supplemented with Matrigel.
　マトリゲル（登録商標　ＢＤ Ｂｉｏｓｃｉｅｎｃｅ社製ＢＤマトリゲルグロースファクターリデュースト）を０～５．０ｖｏｌ％濃度となるよう培地（ＳＣＩＶＡＸ社製 ＮＣＭ－ＭまたはＳＩＧＭＡ社製
 ＲＰＭＩ１６４０）に添加し、当該培地中でＰａｎｃ－１細胞（ＲＣＢ２０９５：ヒト膵癌細胞株
）を７日間培養した。培養は、Ｐａｎｃ－１細胞を１×１０４ｃｅｌｌｓ／１００μｌ／ｗｅｌｌで９６ｗｅｌｌプレートに播種し、３７℃、５％ ＣＯ２条件下にて行った。９６ｗｅｌｌプレートとしては、細胞接着面上に凹凸構造を有したプレート（ＳＣＩＶＡＸ社製 ＮａｎｏＣｕｌｔｕｒｅプレート：凹凸構造面の材質＝環状オレフィン重合体（ＣＯＰ）、凹凸構造の平面形状＝正六角形、単位構造間の幅（線幅）＝７００ｎｍ、単位構造の最小内径＝３μｍ、深さ＝１μｍ）を用いた。 Matrigel (registered trademark, BD Bioscience, BD Matrigel Growth Factor Reduced) is added to a medium (SCIVAX NCM-M or SIGMA RPMI1640) to a concentration of 0 to 5.0 vol%, and Panc- One cell (RCB2095: human pancreatic cancer cell line) was cultured for 7 days. Cultivation was performed by seeding Panc-1 cells in 96-well plates at 1 × 10 4 cells / 100 μl / well and under 37 ° C. and 5% CO 2 conditions. The 96-well plate is a plate having a concavo-convex structure on the cell adhesion surface (NanoCulture plate manufactured by SCIVAX: material of the concavo-convex structure surface = cyclic olefin polymer (COP), planar shape of the concavo-convex structure = regular hexagon, between unit structures The width (line width) = 700 nm, the minimum inner diameter of the unit structure = 3 μm, and the depth = 1 μm were used.
　培養７日目に、各マトリゲル濃度におけるスフェロイドの形成能を光学顕微鏡にて観察した。光学顕微鏡写真を図７に示す。 On the seventh day of culture, the ability to form spheroids at each Matrigel concentration was observed with an optical microscope. An optical micrograph is shown in FIG.
　ＮＣＭ培地では、マトリゲルを添加していない（マトリゲル濃度０ｖｏｌ％）時にはスフェロイドが形成されず、マトリゲル濃度１．０％～５．０ｖｏｌ％でスフェロイドが形成され、特にマトリゲル２．０％～５．０ｖｏｌ％の時に大きなスフェロイドが形成された。 In the NCM medium, when no matrigel is added (matrigel concentration 0 vol%), spheroids are not formed, and spheroids are formed at a matrigel concentration of 1.0% to 5.0 vol%, and in particular, Matrigel 2.0% to 5.0 vol. %, Large spheroids were formed.
　一方、ＲＰＭＩ培地では、マトリゲルを添加していない（マトリゲル濃度０ｖｏｌ％）時に小さなスフェロイドが形成され、これに対してマトリゲル濃度０．５～５．０ｖｏｌ％の時にスフェロイドの数・大きさが向上した。特にマトリゲル濃度１．０ｖｏｌ％の時に良好なスフェロイドが形成された。 On the other hand, in the RPMI medium, small spheroids were formed when no Matrigel was added (Matrigel concentration 0 vol%), whereas the number and size of spheroids were improved when the Matrigel concentration was 0.5 to 5.0 vol%. . In particular, good spheroids were formed when the matrigel concentration was 1.0 vol%.
　マトリゲル（登録商標　ＢＤ Ｂｉｏｓｃｉｅｎｃｅ社製ＢＤマトリゲルグロースファクターリデュースト）を０～５．０ｖｏｌ％濃度となるよう培地（ＳＣＩＶＡＸ社製 ＮＣＭ－ＭまたはＳＩＧＭＡ社製 ＲＰＭＩ１６４０）に添加し、当該培地中でＢｘＰＣ－３細胞（ＣＲＬ－１６８７：ヒト膵癌細胞株）
を７日間培養した。培養は、ＢｘＰＣ－３細胞を１×１０４ｃｅｌｌｓ／１００μｌ／ｗｅｌｌで９６ｗｅｌｌプレートに播種し、３７℃、５％ ＣＯ２条件下にて行った。９６ｗｅｌｌプレートとしては、細胞接着面上に凹凸構造を有したプレート（ＳＣＩＶＡＸ社製 ＮａｎｏＣｕｌｔｕｒｅプレート：凹凸構造面の材質＝環状オレフィン重合体（ＣＯＰ）、凹凸構造の平面形状＝正四角形、単位構造間の幅（線幅）＝７００ｎｍ、単位構造の最小内径＝３μｍ、深さ＝１μｍ）を用いた。 Matrigel (registered trademark BD Bioscience BD Matrigel Growth Factor Reduced) is added to a medium (SCIVAX NCM-M or SIGMA RPMI 1640) to a concentration of 0 to 5.0 vol%, and BxPC- 3 cells (CRL-1687: human pancreatic cancer cell line)
Was cultured for 7 days. Culturing was performed by seeding BxPC-3 cells at 1 × 10 4 cells / 100 μl / well in a 96-well plate at 37 ° C. and 5% CO 2 . The 96-well plate is a plate having a concavo-convex structure on the cell adhesion surface (NanoCulture plate manufactured by SCIVAX: material of the concavo-convex structure surface = cyclic olefin polymer (COP), planar shape of the concavo-convex structure = rectangular, between unit structures The width (line width) = 700 nm, the minimum inner diameter of the unit structure = 3 μm, and the depth = 1 μm were used.
　培養７日目に、各マトリゲル濃度におけるスフェロイドの形成能を光学顕微鏡にて観察した。光学顕微鏡写真を図８に示す。 On the seventh day of culture, the ability to form spheroids at each Matrigel concentration was observed with an optical microscope. An optical micrograph is shown in FIG.
　ＮＣＭ培地では、マトリゲルを添加していない（マトリゲル濃度０ｖｏｌ％）時に小さなスフェロイドが形成された。これに対してマトリゲル濃度1．０～５．０ｖｏｌ％の時にスフェロイドの数・大きさが向上した。特にマトリゲル濃度１％の時に良好なスフェロイドが形成された。 In the NCM medium, small spheroids were formed when no matrigel was added (matrigel concentration 0 vol%). In contrast, the number and size of spheroids improved when the Matrigel concentration was 1.0 to 5.0 vol%. In particular, good spheroids were formed when the matrigel concentration was 1%.
　一方、ＲＰＭＩ培地では、マトリゲルを添加していない（マトリゲル濃度０ｖｏｌ％）時に小さなスフェロイドが形成された。これに対してマトリゲル濃度０．５～５．０ｖｏｌ％の時にスフェロイドの数・大きさが向上した。特にマトリゲル濃度１．０ｖｏｌ％の時に良好なスフェロイドが形成された。また、マトリゲル濃度２．０～５．０ｖｏｌ％の時にはスフェロイドの大きさにバラツキが見られた。 On the other hand, in the RPMI medium, small spheroids were formed when no Matrigel was added (Matrigel concentration 0 vol%). In contrast, the number and size of spheroids improved when the Matrigel concentration was 0.5 to 5.0 vol%. In particular, good spheroids were formed when the matrigel concentration was 1.0 vol%. In addition, when the Matrigel concentration was 2.0 to 5.0 vol%, the spheroid size was varied.
　マトリゲル（登録商標　ＢＤ Ｂｉｏｓｃｉｅｎｃｅ社製ＢＤマトリゲルグロースファクターリデュースト）を０～５．０ｖｏｌ％濃度となるよう培地（ＳＣＩＶＡＸ社製 ＮＣＭ－ＭまたはＳＩＧＭＡ社製 ＲＰＭＩ１６４０）に添加し、当該培地中でｃｏｌｏ２０５細胞（ＥＣ－８７０６１２０８：ヒト大腸癌細胞株）
を７日間培養した。培養は、ｃｏｌｏ２０５細胞を１×１０４ｃｅｌｌｓ／１００μｌ／ｗｅｌｌで９６ｗｅｌｌプレートに播種し、３７℃、５％ ＣＯ２条件下にて行った。９６ｗｅｌｌプレートとしては、細胞接着面上に凹凸構造を有したプレート（ＳＣＩＶＡＸ社製 ＮａｎｏＣｕｌｔｕｒｅプレート：凹凸構造面の材質＝環状オレフィン重合体（ＣＯＰ）、凹凸構造の平面形状＝正六角形、単位構造間の幅（線幅）＝７００ｎｍ、単位構造の最小内径＝３μｍ、深さ＝１μｍ）を用いた。 Matrigel (registered trademark, BD Bioscience, BD Matrigel Growth Factor Reduce) was added to a medium (SCIVAX NCM-M or SIGMA RPMI 1640) to a concentration of 0 to 5.0 vol%, and colo205 cells were added to the medium. (EC-87061208: Human colorectal cancer cell line)
Was cultured for 7 days. Colo205 cells were seeded on a 96-well plate at 1 × 10 4 cells / 100 μl / well and cultured under conditions of 37 ° C. and 5% CO 2 . The 96-well plate is a plate having a concavo-convex structure on the cell adhesion surface (NanoCulture plate manufactured by SCIVAX: material of the concavo-convex structure surface = cyclic olefin polymer (COP), planar shape of the concavo-convex structure = regular hexagon, between unit structures The width (line width) = 700 nm, the minimum inner diameter of the unit structure = 3 μm, and the depth = 1 μm were used.
　培養７日目に、各マトリゲル濃度におけるスフェロイドの形成能を光学顕微鏡にて観察した。光学顕微鏡写真を図９に示す。 On the seventh day of culture, the ability to form spheroids at each Matrigel concentration was observed with an optical microscope. An optical micrograph is shown in FIG.
　ＮＣＭ培地、ＲＰＭＩ培地共に、マトリゲルを添加していない（マトリゲル濃度０ｖｏｌ％）時にはスフェロイドが形成されず、マトリゲル濃度０．５～５．０ｖｏｌ％の時にスフェロイドが形成され、特にマトリゲル濃度１．０ｖｏｌ％の時に良好なスフェロイドが形成された。ＮＣＭ培地では形の整ったスフェロイドが形成され、ＲＰＭＩ培地では大きなスフェロイドが形成された。 In both NCM medium and RPMI medium, spheroids are not formed when no matrigel is added (matrigel concentration 0 vol%), and spheroids are formed when the matrigel concentration is 0.5 to 5.0 vol%, and in particular, the matrigel concentration is 1.0 vol%. At that time, good spheroids were formed. NCM medium formed well-shaped spheroids, and RPMI medium formed large spheroids.
　マトリゲル（登録商標　ＢＤ Ｂｉｏｓｃｉｅｎｃｅ社製ＢＤマトリゲルグロースファクターリデュースト）を０～５．０ｖｏｌ％濃度となるよう培地（ＳＣＩＶＡＸ社製 ＮＣＭ－ＭまたはＳＩＧＭＡ社製 Ｌ－１５
）に添加し、当該培地中でＭＤＡ－ＭＢ－２３１細胞（ＨＴＢ－２６：ヒト乳癌細胞株）
を７日間培養した。培養は、ＭＤＡ－ＭＢ－２３１細胞を１×１０４ｃｅｌｌｓ／１００μｌ／ｗｅｌｌで９６ｗｅｌｌプレートに播種し、３７℃、５％ ＣＯ２条件下にて行った。９６ｗｅｌｌプレートとしては、細胞接着面上に凹凸構造を有したプレート（ＳＣＩＶＡＸ社製 ＮａｎｏＣｕｌｔｕｒｅプレート：凹凸構造面の材質＝環状オレフィン重合体（ＣＯＰ）、凹凸構造の平面形状＝正六角形、単位構造間の幅（線幅）＝７００ｎｍ、単位構造の最小内径＝３μｍ、深さ＝１μｍ）を用いた。 Matrigel (registered trademark BD Bioscience's BD Matrigel Growth Factor Reduce) medium (SCIVAX's NCM-M or SIGMA's L-15) at a concentration of 0 to 5.0 vol%
MDA-MB-231 cells (HTB-26: human breast cancer cell line)
Was cultured for 7 days. Culturing was performed by seeding MDA-MB-231 cells in 96-well plates at 1 × 10 4 cells / 100 μl / well at 37 ° C. under 5% CO 2 conditions. The 96-well plate is a plate having a concavo-convex structure on the cell adhesion surface (NanoCulture plate manufactured by SCIVAX: material of the concavo-convex structure surface = cyclic olefin polymer (COP), planar shape of the concavo-convex structure = regular hexagon, between unit structures The width (line width) = 700 nm, the minimum inner diameter of the unit structure = 3 μm, and the depth = 1 μm were used.
　培養７日目に、各マトリゲル濃度におけるスフェロイドの形成能を光学顕微鏡にて観察した。光学顕微鏡写真を図１０に示す。 On the seventh day of culture, the ability to form spheroids at each Matrigel concentration was observed with an optical microscope. An optical micrograph is shown in FIG.
　ＮＣＭ培地、Ｌ－１５培地共に、マトリゲルを添加していない（マトリゲル濃度０ｖｏｌ％）時には小さなスフェロイドが形成され、マトリゲル濃度１．０～５．０ｖｏｌ％の時に、より大きなスフェロイドが形成された。ＮＣＭ培地では、マトリゲル濃度５．０ｖｏｌ％の時に形の整った良好なスフェロイドが形成され、Ｌ－１５培地では、マトリゲル濃度２．０ｖｏｌ％の時に大きなスフェロイドが形成された。 In both NCM medium and L-15 medium, small spheroids were formed when no matrigel was added (matrigel concentration 0 vol%), and larger spheroids were formed when the matrigel concentration was 1.0 to 5.0 vol%. In the NCM medium, good shaped spheroids were formed when the Matrigel concentration was 5.0 vol%, and in the L-15 medium, large spheroids were formed when the Matrigel concentration was 2.0 vol%.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
　マトリゲル（登録商標　ＢＤ Ｂｉｏｓｃｉｅｎｃｅ社製　ＢＤマトリゲルグロースファクターリデュースト　フェノールレッドフリー）又はその構成成分であるＩＶ型コラーゲン（ＢＤ Ｂｉｏｓｃｉｅｎｃｅ社製　コラーゲンＩＶ（マウス））、ラミニン（ＢＤ Ｂｉｏｓｃｉｅｎｃｅ社製　ラミニン（マウス））、ラミニン／エンタクチン（ＢＤ Ｂｉｏｓｃｉｅｎｃｅ社製　高濃度ラミニン／エンタクチン複合体（マウス））を各々表１の濃度となるように培地（ＳＣＩＶＡＸ社製 ＮＣＭ－Ｍ）に添加し、当該培地中でＰａｎｃ－１細胞（ＲＣＢ２０９５：ヒト膵癌細胞株
）を７日間培養した。培養は、Ｐａｎｃ－１細胞を１×１０４ｃｅｌｌｓ／１００μｌ／ｗｅｌｌで９６ｗｅｌｌプレートに播種し、３７℃、５％ ＣＯ２条件下にて行った。９６ｗｅｌｌプレートとしては、細胞接着面上に凹凸構造を有したプレート（ＳＣＩＶＡＸ社製 ＮａｎｏＣｕｌｔｕｒｅプレート：凹凸構造面の材質＝環状オレフィン重合体（ＣＯＰ）、凹凸構造の平面形状＝正六角形、単位構造間の幅（線幅）＝７００ｎｍ、単位構造の最小内径＝３μｍ、深さ＝１μｍ）を用いた。 Matrigel (registered trademark BD Bioscience manufactured by BD Matrigel Growth Factor Reduced Phenol Red Free) or its type IV collagen (Collagen IV (mouse) manufactured by BD Bioscience), laminin (Laminin manufactured by BD Bioscience) Laminin / entactin (high concentration laminin / entactin complex (mouse) manufactured by BD Bioscience) was added to the culture medium (SCMAX NCM-M) at the concentration shown in Table 1, and Panc-1 Cells (RCB2095: human pancreatic cancer cell line) were cultured for 7 days. Cultivation was performed by seeding Panc-1 cells in 96-well plates at 1 × 10 4 cells / 100 μl / well and under 37 ° C. and 5% CO 2 conditions. The 96-well plate is a plate having a concavo-convex structure on the cell adhesion surface (NanoCulture plate manufactured by SCIVAX: material of the concavo-convex structure surface = cyclic olefin polymer (COP), planar shape of the concavo-convex structure = regular hexagon, between unit structures The width (line width) = 700 nm, the minimum inner diameter of the unit structure = 3 μm, and the depth = 1 μm were used.
　培養７日目に、スフェロイドの形成能を光学顕微鏡にて観察した。光学顕微鏡写真を図１１に示す。 On the seventh day of culture, spheroid formation ability was observed with an optical microscope. An optical micrograph is shown in FIG.
　マトリゲル構成成分を添加していない培地（無添加）ではほとんどスフェロイドが形成されなかったのに対し、ＩＶ型コラーゲン又はラミニン又はラミニン／エンタクチン複合体を添加した培地では小さなスフェロイドが形成された。ＩＶ型コラーゲンとラミニン／エンタクチン複合体を添加した培地では大きなスフェロイドが形成された。このスフェロイド形成能は、ほぼ同濃度のマトリゲル添加時と同程度であった。 Spheroids were hardly formed in the medium to which no Matrigel component was added (no addition), whereas small spheroids were formed in the medium to which type IV collagen or laminin or laminin / entactin complex was added. Large spheroids were formed in the medium supplemented with type IV collagen and laminin / entactin complex. This spheroid-forming ability was almost the same as when adding the same concentration of matrigel.
　マトリゲル（登録商標　ＢＤ Ｂｉｏｓｃｉｅｎｃｅ社製　ＢＤマトリゲルグロースファクターリデュースト　フェノールレッドフリー）又はその構成成分であるＩＶ型コラーゲン（ＢＤ Ｂｉｏｓｃｉｅｎｃｅ社製　コラーゲンＩＶ（マウス））、ラミニン（ＢＤ Ｂｉｏｓｃｉｅｎｃｅ社製　ラミニン（マウス））、ラミニン／エンタクチン（ＢＤ Ｂｉｏｓｃｉｅｎｃｅ社製　高濃度ラミニン／エンタクチン複合体（マウス））を各々表１の濃度となるように培地（ＳＣＩＶＡＸ社製 ＮＣＭ－Ｍ）に添加し、当該培地中でＢｘＰＣ－３細胞（ＣＲＬ－１６８７：ヒト膵癌細胞株）
を７日間培養した。培養は、ＢｘＰＣ－３細胞を１×１０４ｃｅｌｌｓ／１００μｌ／ｗｅｌｌで９６ｗｅｌｌプレートに播種し、３７℃、５％ ＣＯ２条件下にて行った。９６ｗｅｌｌプレートとしては、細胞接着面上に凹凸構造を有したプレート（ＳＣＩＶＡＸ社製 ＮａｎｏＣｕｌｔｕｒｅプレート：凹凸構造面の材質＝環状オレフィン重合体（ＣＯＰ）、凹凸構造の平面形状＝正四角形、単位構造間の幅（線幅）＝７００ｎｍ、単位構造の最小内径＝３μｍ、深さ＝１μｍ）を用いた。 Matrigel (registered trademark BD Bioscience manufactured by BD Matrigel Growth Factor Reduced Phenol Red Free) or its type IV collagen (Collagen IV (mouse) manufactured by BD Bioscience), laminin (Laminin manufactured by BD Bioscience) Laminin / entactin (BD Bioscience's high-concentration laminin / entactin complex (mouse)) was added to the medium (SCVAX NCM-M) at the concentrations shown in Table 1, and BxPC-3 was added in the medium. Cells (CRL-1687: human pancreatic cancer cell line)
Was cultured for 7 days. Culturing was performed by seeding BxPC-3 cells at 1 × 10 4 cells / 100 μl / well in a 96-well plate at 37 ° C. and 5% CO 2 . The 96-well plate is a plate having a concavo-convex structure on the cell adhesion surface (NanoCulture plate manufactured by SCIVAX: material of the concavo-convex structure surface = cyclic olefin polymer (COP), planar shape of the concavo-convex structure = rectangular, between unit structures The width (line width) = 700 nm, the minimum inner diameter of the unit structure = 3 μm, and the depth = 1 μm were used.
　培養７日目に、スフェロイドの形成能を光学顕微鏡にて観察した。光学顕微鏡写真を図１２に示す。 On the seventh day of culture, spheroid formation ability was observed with an optical microscope. An optical micrograph is shown in FIG.
　マトリゲル構成成分を添加していない培地（無添加）では小さなスフェロイドが形成された。これに対して、ＩＶ型コラーゲン又はラミニン又はラミニン／エンタクチン複合体を添加した培地では、スフェロイドの数・大きさが向上した。ＩＶ型コラーゲンとラミニン／エンタクチン複合体を添加した培地では、同濃度のマトリゲル添加時と同程度の大きなスフェロイドが形成された。 Small spheroids were formed in the medium not added with Matrigel component (no addition). In contrast, the number and size of spheroids improved in the medium supplemented with type IV collagen, laminin, or laminin / entactin complex. In the medium supplemented with type IV collagen and laminin / entactin complex, large spheroids were formed which were similar to those obtained when the same concentration of matrigel was added.
　マトリゲル（登録商標　ＢＤ Ｂｉｏｓｃｉｅｎｃｅ社製　ＢＤマトリゲルグロースファクターリデュースト　フェノールレッドフリー）又はその構成成分であるＩＶ型コラーゲン（ＢＤ Ｂｉｏｓｃｉｅｎｃｅ社製　コラーゲンＩＶ（マウス））、ラミニン（ＢＤ Ｂｉｏｓｃｉｅｎｃｅ社製　ラミニン（マウス））、ラミニン／エンタクチン（ＢＤ Ｂｉｏｓｃｉｅｎｃｅ社製　高濃度ラミニン／エンタクチン複合体（マウス））を各々表１の濃度となるように培地（ＳＣＩＶＡＸ社製 ＮＣＭ－Ｍ）に添加し、当該培地中でｃｏｌｏ２０５細胞（ＥＣ－８７０６１２０８：ヒト大腸癌細胞株）
を７日間培養した。培養は、ｃｏｌｏ２０５細胞を１×１０４ｃｅｌｌｓ／１００μｌ／ｗｅｌｌで９６ｗｅｌｌプレートに播種し、３７℃、５％ ＣＯ２条件下にて行った。９６ｗｅｌｌプレートとしては、細胞接着面上に凹凸構造を有したプレート（ＳＣＩＶＡＸ社製 ＮａｎｏＣｕｌｔｕｒｅプレート：凹凸構造面の材質＝環状オレフィン重合体（ＣＯＰ）、凹凸構造の平面形状＝正六角形、単位構造間の幅（線幅）＝７００ｎｍ、単位構造の最小内径＝３μｍ、深さ＝１μｍ）を用いた。 Matrigel (registered trademark BD Bioscience manufactured by BD Matrigel Growth Factor Reduced Phenol Red Free) or its type IV collagen (Collagen IV (mouse) manufactured by BD Bioscience), laminin (Laminin manufactured by BD Bioscience) Laminin / entactin (BD Bioscience's high-concentration laminin / entactin complex (mouse)) was added to the medium (SCIVAX NCM-M) at the concentrations shown in Table 1 and colo205 cells ( EC-87061208: Human colorectal cancer cell line)
Was cultured for 7 days. Colo205 cells were seeded on a 96-well plate at 1 × 10 4 cells / 100 μl / well and cultured under conditions of 37 ° C. and 5% CO 2 . The 96-well plate is a plate having a concavo-convex structure on the cell adhesion surface (NanoCulture plate manufactured by SCIVAX: material of the concavo-convex structure surface = cyclic olefin polymer (COP), planar shape of the concavo-convex structure = regular hexagon, between unit structures The width (line width) = 700 nm, the minimum inner diameter of the unit structure = 3 μm, and the depth = 1 μm were used.
　培養７日目に、各マトリゲル濃度におけるスフェロイドの形成能を光学顕微鏡にて観察した。光学顕微鏡写真を図１３に示す。 On the seventh day of culture, the ability to form spheroids at each Matrigel concentration was observed with an optical microscope. An optical micrograph is shown in FIG.
　マトリゲル構成成分を添加していない培地（無添加）ではスフェロイドが形成されなかったのに対して、ＩＶ型コラーゲン又はラミニン又はラミニン／エンタクチン複合体を添加した培地では、小さなスフェロイドが形成された。ＩＶ型コラーゲンとラミニン／エンタクチン複合体を添加した培地では、さらにスフェロイドの大きさが向上した。 Spheroids were not formed in the medium to which no Matrigel component was added (no addition), whereas small spheroids were formed in the medium to which type IV collagen or laminin or laminin / entactin complex was added. In the medium supplemented with type IV collagen and laminin / entactin complex, the size of spheroids was further improved.
本発明は、優れたスフェロイドの形成を可能とするため、再生医療、細胞工学、組織工学などの医療・バイオテクノロジーの分野に関わる広範な技術に使用でき、薬剤のスクリーニングや毒性評価をはじめ、医薬品、化粧品、食品の有効性・機能性評価、安全性評価等に有用である。 Since the present invention enables the formation of excellent spheroids, it can be used in a wide range of technologies related to the medical and biotechnology fields such as regenerative medicine, cell engineering, tissue engineering, pharmaceutical screening and toxicity evaluation, It is useful for evaluating the effectiveness and functionality of cosmetics and foods, and evaluating safety.
１　単位構造
２　線 1 unit structure 2 lines

Claims (16)

  1. 　細胞接着面として機能する凹凸構造を有する培養基材上において、
    　培地基礎成分と、培養温度にてゲル化しない濃度の細胞外マトリックス成分との存在下で、
    細胞を培養することを特徴とするスフェロイド形成促進方法。     On a culture substrate having a concavo-convex structure that functions as a cell adhesion surface,
    In the presence of medium basic components and extracellular matrix components at a concentration that does not gel at the culture temperature,
    A method for promoting spheroid formation, comprising culturing cells.
  2. 　前記凹凸構造は、単位構造間の幅が３μｍ以下で平面方向の形状が多角形であると共に最小内径が３μｍ以下の単位構造を規則的に複数配列して形成されていることを特徴とする請求項１記載のスフェロイド形成促進方法。 The concavo-convex structure is formed by regularly arranging a plurality of unit structures having a width between unit structures of 3 μm or less, a planar shape of a polygon and a minimum inner diameter of 3 μm or less. Item 4. The method for promoting spheroid formation according to Item 1.
  3. 　前記細胞外マトリックス成分の含有量が０．０５ｍｇ／ｍｌ以下である請求項１又は２いずれか記載のスフェロイド形成促進方法。 The method for promoting spheroid formation according to claim 1 or 2, wherein the content of the extracellular matrix component is 0.05 mg / ml or less.
  4. 　前記細胞外マトリックス成分の含有量が０．０２ｍｇ／ｍｌ以下である請求項１又は２いずれか記載のスフェロイド形成促進方法。 The method for promoting spheroid formation according to claim 1 or 2, wherein the content of the extracellular matrix component is 0.02 mg / ml or less.
  5. 　前記細胞外マトリックス成分の含有量が０．０１ｍｇ／ｍｌ以下である請求項１又は２いずれか記載のスフェロイド形成促進方法。 The method for promoting spheroid formation according to claim 1 or 2, wherein the content of the extracellular matrix component is 0.01 mg / ml or less.
  6. 　前記細胞外マトリックス成分が基底膜成分であることを特徴とする請求項１乃至５いずれか記載のスフェロイド形成促進方法。 The method for promoting spheroid formation according to any one of claims 1 to 5, wherein the extracellular matrix component is a basement membrane component.
  7. 　前記細胞外マトリックス成分が少なくともラミニン、ＩＶ型コラーゲン、ラミニン／エンタクチン複合体から選択される１以上の成分を含むことを特徴とする請求項１乃至５いずれか記載のスフェロイド形成促進方法。 The method for promoting spheroid formation according to any one of claims 1 to 5, wherein the extracellular matrix component contains at least one component selected from laminin, type IV collagen, and laminin / entactin complex.
  8. 　前記細胞外マトリックス成分がＥｎｇｅｌｂｒｅｔｈ－Ｈｏｌｍ－Ｓｗａｒｍ（ＥＨＳ）マウス腫瘍からの抽出物であることを特徴とする請求項１乃至５いずれか記載のスフェロイド形成促進方法。 6. The method for promoting spheroid formation according to any one of claims 1 to 5, wherein the extracellular matrix component is an extract from an Engelbreth-Holm-Swarm (EHS) mouse tumor.
  9. 　細胞接着面として機能する凹凸構造上の細胞に用いられるものであって、培地基礎成分と、培養温度にてゲル化しない濃度の細胞外マトリックス成分とを含有することを特徴とするスフェロイド形成促進培地。 A spheroid formation-promoting medium that is used for cells on a concavo-convex structure that functions as a cell adhesion surface, and includes a medium basic component and an extracellular matrix component at a concentration that does not gel at the culture temperature. .
  10. 　単位構造間の幅が３μｍ以下で平面方向の形状が多角形であると共に最小内径が３μｍ以下の単位構造を規則的に複数配列して形成された前記凹凸構造上の細胞に用いられることを特徴とする請求項９記載のスフェロイド形成促進培地。 It is used for cells on the concavo-convex structure formed by regularly arranging a plurality of unit structures whose width between unit structures is 3 μm or less and whose planar shape is a polygon and whose minimum inner diameter is 3 μm or less. The spheroid formation promoting medium according to claim 9.
  11. 　前記細胞外マトリックス成分の含有量が０．０５ｍｇ／ｍｌ以下である請求項９又は１０いずれか記載のスフェロイド形成促進培地。 The spheroid formation promoting medium according to claim 9 or 10, wherein the content of the extracellular matrix component is 0.05 mg / ml or less.
  12. 　前記細胞外マトリックス成分の含有量が０．０２ｍｇ／ｍｌ以下である請求項９又は１０いずれか記載のスフェロイド形成促進培地。 The spheroid formation promotion medium according to claim 9 or 10, wherein the content of the extracellular matrix component is 0.02 mg / ml or less.
  13. 　前記細胞外マトリックス成分の含有量が０．０１ｍｇ／ｍｌ以下である請求項９又は１０いずれか記載のスフェロイド形成促進培地。 The medium for promoting spheroid formation according to claim 9 or 10, wherein the content of the extracellular matrix component is 0.01 mg / ml or less.
  14. 　前記細胞外マトリックス成分が基底膜成分であることを特徴とする請求項９乃至１３いずれか記載のスフェロイド形成促進培地。 The spheroid formation promoting medium according to any one of claims 9 to 13, wherein the extracellular matrix component is a basement membrane component.
  15. 　前記細胞外マトリックス成分が少なくともラミニン、ＩＶ型コラーゲン、ラミニン／エンタクチン複合体から選択される１以上の成分を含むことを特徴とする請求項９乃至１３いずれか記載のスフェロイド形成促進培地。 The spheroid formation promoting medium according to any one of claims 9 to 13, wherein the extracellular matrix component contains at least one component selected from laminin, type IV collagen, and laminin / entactin complex.
  16. 　前記細胞外マトリックス成分がＥｎｇｅｌｂｒｅｔｈ－Ｈｏｌｍ－Ｓｗａｒｍ（ＥＨＳ）マウス腫瘍からの抽出物であることを特徴とする請求項９乃至１３いずれか記載のスフェロイド形成促進培地。 The spheroid formation promoting medium according to any one of claims 9 to 13, wherein the extracellular matrix component is an extract from an Engelbreth-Holm-Swarm (EHS) mouse tumor.
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