WO2011077894A1 - Procédé et milieu de culture permettant d'améliorer la formation d'un sphéroïde - Google Patents

Procédé et milieu de culture permettant d'améliorer la formation d'un sphéroïde 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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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

Cette invention concerne un procédé de culture cellulaire capable de former un sphéroïde par des procédés simples à court terme. Un milieu de culture permettant d'améliorer la formation d'un sphéroïde qui peut être utilisé dans ledit procédé de culture cellulaire est également décrit. La culture des cellules sur un substrat de culture ayant une structure en creux et projections qui opère comme une surface d'adhérence cellulaire et l'utilisation d'un milieu de culture cellulaire qui contient un composant de matrice extracellulaire prédéterminé empêchent la prolifération et la croissance de monocouches cellulaires ainsi que la séparation et l'affaissement d'un sphéroïde, améliorant ainsi d'une manière extrêmement efficace la formation du sphéroïde.
PCT/JP2010/071002 2009-12-25 2010-11-25 Procédé et milieu de culture permettant d'améliorer la formation d'un sphéroïde WO2011077894A1 (fr)

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WO2017146122A1 (fr) * 2016-02-22 2017-08-31 凸版印刷株式会社 Procédé pour promouvoir la formation de sphéroïdes
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WO2018169007A1 (fr) * 2017-03-16 2018-09-20 株式会社Ｌｓｉメディエンス Culture tridimensionnelle de cellules cancéreuses primaires utilisant un tissu tumoral

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Cited By (12)

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JP2013223442A (ja) * 2012-04-20 2013-10-31 Dainippon Printing Co Ltd 細胞遊走解析方法および細胞培養物作成方法
US20180245050A1 (en) * 2015-09-03 2018-08-30 The Brigham And Women's Hospital, Inc. Three-dimensional differentiation of epiblast spheroids to kidney organoids models stage-specific epithelial physiology, morphogenesis, and disease
US10815460B2 (en) * 2015-09-03 2020-10-27 The Brigham And Women's Hospital, Inc. Three-dimensional differentiation of epiblast spheroids to kidney organoids models stage-specific epithelial physiology, morphogenesis, and disease
WO2017146122A1 (fr) * 2016-02-22 2017-08-31 凸版印刷株式会社 Procédé pour promouvoir la formation de sphéroïdes
JP2017147944A (ja) * 2016-02-22 2017-08-31 凸版印刷株式会社 スフェロイド形成促進方法
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WO2018169007A1 (fr) * 2017-03-16 2018-09-20 株式会社Ｌｓｉメディエンス Culture tridimensionnelle de cellules cancéreuses primaires utilisant un tissu tumoral
CN110475860A (zh) * 2017-03-16 2019-11-19 美迪恩斯生命科技株式会社 使用肿瘤组织的原代癌细胞的三维培养
JPWO2018169007A1 (ja) * 2017-03-16 2020-02-13 株式会社Ｌｓｉメディエンス 腫瘍組織を用いた初代がん細胞の３次元培養
US11549100B2 (en) 2017-03-16 2023-01-10 Lsi Medience Corporation Three-dimensional culture of primary cancer cells using tumor tissue
US11873514B2 (en) 2017-03-16 2024-01-16 Lsi Medience Corporation Method of screening for a substance that acts on a cell mass
CN110475860B (zh) * 2017-03-16 2024-05-14 美迪恩斯生命科技株式会社 使用肿瘤组织的原代癌细胞的三维培养

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