WO2023085019A1 - 培養容器及び培養方法 - Google Patents

培養容器及び培養方法 Download PDF

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Publication number
WO2023085019A1
WO2023085019A1 PCT/JP2022/038875 JP2022038875W WO2023085019A1 WO 2023085019 A1 WO2023085019 A1 WO 2023085019A1 JP 2022038875 W JP2022038875 W JP 2022038875W WO 2023085019 A1 WO2023085019 A1 WO 2023085019A1
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Prior art keywords
culture vessel
oxygen
gas barrier
barrier layer
culture
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PCT/JP2022/038875
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English (en)
French (fr)
Japanese (ja)
Inventor
勝弘 江刺家
寛 宮廻
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Mitsui Chemicals Inc
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Mitsui Chemicals Inc
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Priority to CN202280073411.6A priority Critical patent/CN118176290A/zh
Priority to JP2023527720A priority patent/JP7364825B2/ja
Priority to EP22892519.4A priority patent/EP4431592A4/en
Publication of WO2023085019A1 publication Critical patent/WO2023085019A1/ja
Priority to JP2023173465A priority patent/JP2024001173A/ja
Anticipated expiration legal-status Critical
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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
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/02—Form or structure of the vessel
    • C12M23/12—Well or multiwell plates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/20—Material Coatings
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/22—Transparent or translucent parts
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/24—Gas permeable parts

Definitions

  • One aspect of the present invention relates to a culture vessel or culture method.
  • Cells, tissues, or organs (hereinafter also referred to as cells, etc.) cannot be cultured unless they are grown under conditions suitable for their growth.
  • the container must be placed in an incubator capable of keeping the temperature, humidity and gas concentration at predetermined levels. Furthermore, sufficient and appropriate oxygen supply must be performed in order to efficiently realize the above culture.
  • Non-Patent Document 1 uses polydimethylsiloxane (PDMS) on the bottom surface of the culture vessel to culture hepatocytes with a high oxygen consumption rate, thereby eliminating the oxygen-deficient state seen in commercially available polystyrene plates. They reported that a high degree of self-organization of hepatocytes was observed. Further, Patent Document 1 discloses a cell, tissue or organ culture material containing a 4-methyl-1-pentene polymer and having excellent oxygen supply properties.
  • PDMS polydimethylsiloxane
  • one aspect of the present invention provides a culture vessel or a culture method in which cells and the like can easily adhere to the culture vessel and the functions of the cells and the like can be maintained normally.
  • a culture vessel for culturing cells, tissues, or organs in a medium wherein at least part of the culture surface of the culture vessel has an oxygen-permeable layer (I- a) and a gas barrier layer (II-a) satisfying the following requirements (3) and (4), wherein the gas barrier layer (II-a) is the lower surface of the oxygen permeable layer (Ia) wherein the gas barrier layer (II-a) is detachable from the oxygen permeable layer (Ia) during culture.
  • the oxygen permeability P (Ia) at a temperature of 23°C and a humidity of 0% is 20,000 to 60,000 cm 3 /(m 2 ⁇ 24h ⁇ atm).
  • the measured total light transmittance is 70% or more.
  • the oxygen permeability P(II-a) at a temperature of 23° C. and a humidity of 0% is 3000 cm 3 /(m 2 ⁇ 24 h ⁇ atm) or less.
  • the total light transmittance measured according to JIS K 7361-1 is 70% or more [2]
  • the water contact angle of the oxygen permeable layer (Ia) is 30 to 120° [1 ].
  • the 4-methyl-1-pentene polymer (X) is a 4-methyl-1-pentene homopolymer (x1), 4-methyl-1-pentene, ethylene and C 3-20
  • the gas barrier layer (II-a) contains polyethylene terephthalate (PET).
  • a method for culturing a cell, tissue, or organ comprising a step (Aa) of culturing the cell, tissue, or organ using the culture vessel according to any one of [1] to [6]. , the step (Ba) of removing the gas barrier layer (II-a) from the oxygen permeable layer (Ia), and using the culture vessel obtained in the step (Ba), cells, tissues, or a step of further culturing the organ (Ca).
  • a culture vessel for culturing cells, tissues, or organs wherein the culture vessel has a bottom member and a side wall member, and the bottom member and the side wall member are joined to form at least one A culture space is formed, and at least part of the bottom member has at least an oxygen permeable layer (Ib) and a gas barrier layer (II-b), and the gas barrier layer (II-b) Under the permeable layer (Ib), the gas barrier layer (II-b) is detachably superimposed from the oxygen permeable layer (Ib), the gas barrier layer (II-b) satisfies the following requirement (5), and the oxygen permeable layer ( A culture vessel in which Ib) satisfies the following requirement (6).
  • the oxygen permeability P(II-b) at a temperature of 23°C and a humidity of 0% is 3000 cm 3 /(m 2 x 24 h x atm) or less.
  • the oxygen permeability P (Ib) is 6.0 times or more the P (II-b) [ii]
  • the gas barrier layer (II-b) is selected from the group consisting of polyethylene terephthalate (PET) and polyolefin and satisfying the following requirements (7) or (8), the culture vessel according to [i].
  • the peel strength of the gas barrier layer (II-b) to the oxygen permeable layer (Ib) measured by the following method is 0.01 to The culture vessel according to [i] or [ii], which is 0.20 N/25 mm.
  • the 4-methyl-1-pentene polymer (X) is a 4-methyl-1-pentene homopolymer (x1), 4-methyl-1-pentene, ethylene and The culture according to [iv], which is at least one polymer selected from copolymers (x2) with at least one olefin selected from ⁇ -olefins (excluding 4-methyl-1-pentene). container. [vi] The culture vessel according to any one of [i] to [v], wherein the P(Ib) is 20,000 to 60,000 cm 3 /(m 2 ⁇ 24h ⁇ atm).
  • the total light transmittance of the region of the bottom member having the oxygen permeable layer (Ib) and the gas barrier layer (II-b) measured according to JIS K 7361-1 is 70%.
  • [xii] A method for culturing a cell, tissue, or organ, comprising the step of culturing the cell, tissue, or organ using the culture vessel according to any one of [i] to [xi] (Ab) , the step (Bb) of removing the gas barrier layer (II-b) from the oxygen permeable layer (Ib), and using the culture vessel obtained in the step (Bb), cells, tissues, or a step (Cb) of further culturing the organ.
  • [xiii] The method for manufacturing a culture vessel according to any one of [i] to [xii], wherein the method includes a step of attaching the bottom member to the side wall member.
  • a culture vessel and a culture method in which cells and the like can easily adhere to the culture vessel and the functions of the cells and the like can be maintained normally.
  • FIG. 1 is a schematic cross-sectional view of a culture vessel.
  • the left side of FIG. 1 is a schematic diagram of the culture surface having the gas barrier layer (II), and the right side of FIG. 1 is a schematic diagram of the state where the gas barrier layer (II) is removed from the oxygen permeable layer (I).
  • 2 is a photograph showing the morphology of human frozen hepatocytes after 24 hours of culture in Example 1.
  • FIG. 3 is a photograph showing the morphology of human frozen hepatocytes after 24 hours of culture in Comparative Example 2.
  • FIG. 1 is a schematic cross-sectional view of a culture vessel.
  • the left side of FIG. 1 is a schematic diagram of the culture surface having the gas barrier layer (II)
  • the right side of FIG. 1 is a schematic diagram of the state where the gas barrier layer (II) is removed from the oxygen permeable layer (I).
  • 2 is a photograph showing the morphology of human frozen hepatocytes after 24 hours of culture in Example
  • One aspect of the present invention is a culture vessel for culturing cells, tissues, or organs in a medium, wherein at least part of the culture surface of the culture vessel comprises an oxygen permeable layer (Ia) that satisfies the following requirements (1) and (2); Having a laminated region with a gas barrier layer (II-a) that satisfies the following requirements (3) and (4), The gas barrier layer (II-a) is provided on the lower surface of the oxygen permeable layer (Ia), The culture vessel is such that the gas barrier layer (II-a) is detachable from the oxygen permeable layer (Ia) during culture.
  • This culture vessel is called a culture vessel ( ⁇ ).
  • the oxygen permeability P (Ia) at a temperature of 23°C and a humidity of 0% is 20,000 to 60,000 cm 3 /(m 2 ⁇ 24h ⁇ atm).
  • the measured total light transmittance is 70% or more.
  • the oxygen permeability P(II-a) at a temperature of 23° C. and a humidity of 0% is 3000 cm 3 /(m 2 ⁇ 24 h ⁇ atm) or less. 4)
  • Total light transmittance measured in accordance with JIS K 7361-1 is 70% or more
  • a culture vessel for culturing cells, tissues, or organs having a bottom member and a side wall member, At least one culture space is formed by joining the bottom member and the side wall member, at least part of the bottom member has at least an oxygen permeable layer (Ib) and a gas barrier layer (II-b),
  • the gas barrier layer (II-b) is superimposed under the oxygen permeable layer (Ib) so as to be detachable from the oxygen permeable layer (Ib),
  • the gas barrier layer (II-b) satisfies the following requirement (5)
  • the culture vessel, wherein the oxygen-permeable layer (Ib) satisfies the following requirement (6).
  • This culture vessel is called a culture vessel ( ⁇ ).
  • the oxygen permeability P(II-b) at a temperature of 23°C and a humidity of 0% is 3000 cm 3 /(m 2 ⁇ 24 h ⁇ atm) or less.
  • the oxygen permeability P(Ib) is at least 6.0 times the P(II-b).
  • cells, tissues, or organs are also simply referred to as "cells, etc.”
  • the origin of cells and the like is not particularly limited, and they may be any organism such as animals, plants, insects, fungi, protozoa, and bacteria. Animals or plants are preferred, animals are more preferred, and mammals are particularly preferred.
  • the culture vessel which is one embodiment of the present invention has excellent adhesion to cells and the like, the cells and the like are preferably adherent.
  • Cells and the like are preferably cells because they are suitable for culturing on the culture surface of the culture vessel.
  • the cells and the like are preferably aerobic, and more preferably do not contain anaerobic cells.
  • a tissue in one aspect of the present invention means a collection of similar cells that perform similar functions.
  • the tissue is not particularly limited, and examples thereof include epithelial tissue, connective tissue, muscle tissue, nerve tissue and the like.
  • the tissue is preferably liver lobule, cardiac muscle tissue, nerve tissue, or skeletal muscle tissue, more preferably liver lobule, because of its high oxygen requirement.
  • An organ in one aspect of the present invention means a group of the above-mentioned tissues that work together with a purpose.
  • the organ is not particularly limited, and examples thereof include lung, heart, liver, kidney, spleen, pancreas, gallbladder, esophagus, stomach, skin, and brain.
  • the organ is preferably the skin, kidney, liver, heart, or brain, more preferably the liver, because of its high oxygen demand.
  • Cells in one aspect of the present invention are not particularly limited, but are preferably animal cells.
  • the cells may be suspension cells or adherent cells, preferably adherent cells.
  • Cells may be two-dimensionally cultured cells or three-dimensionally cultured cells, and include spheroids obtained by culturing cells.
  • the cells may be primary cultured cells or subcultured cells, but primary cultured cells are preferred. Cells may be frozen or refrozen.
  • animal cells normal cells, cancer cells, fusion cells such as hybridomas, etc. can be used, and cells that have undergone artificial treatment such as gene transfer may also be used.
  • animal cells include undifferentiated pluripotent stem cells, pluripotent stem cells in the process of differentiation, differentiated cells derived from pluripotent stem cells, pluripotent stem cells, somatic stem cells, and animal tissue-derived cells. differentiated cells can be used.
  • the animal cells may be derived from any animal, but mammals are preferred, and human, bovine, dog, cat, pig, minipig, rabbit, hamster, rat, or mouse cells are particularly preferred. , rat, mouse or bovine cells are more preferred.
  • the cells in one aspect of the present invention are preferably cells constituting skin, kidney, liver, brain, nerve tissue, cardiac muscle tissue, or skeletal muscle tissue, or cancer cells, more preferably hepatocytes, kidney Cells, cardiomyocytes, nerve cells, more preferably hepatocytes.
  • the cells may be used singly or in combination of two or more.
  • Cells may also include or exclude stem cells or progenitor cells.
  • the cells may exclude or include epithelial stem cells or progenitor cells.
  • Hepatocytes in one aspect of the present invention may be any cell in the liver including hepatocytes, specifically vascular endothelial cells, vascular smooth muscle cells, adipocytes, Including blood cells, hepatic mononuclear cells, hepatic macrophages (including Kupffer cells), hepatic stellate cells, intrahepatic bile duct epithelial cells, and the like.
  • the hepatocytes are, for example, a cell population containing 20% or more, 30% or more, 40% or more, or 50% or more hepatocytes.
  • the hepatocytes may be primary cultured cells or established subcultured cells, but primary cultured cells are preferably used as hepatocytes.
  • the type of subcultured cell line is not particularly limited, but for example, SSP-25, RBE, HepG2, TGBC50TKB, HuH-6, HuH-7, ETK-1, Het-1A, PLC/PRF/5, Hep3B, SK - HEP-1, C3A, THLE-2, THLE-3, HepG2/2.2.1, SNU-398, SNU-449, SNU-182, SNU-475, SNU-387, SNU-423, FL62891, DMS153 etc.
  • the hepatocytes may be a cell population containing cell types other than hepatocytes, for example, a cell population containing 20% or more, 30% or more, 40% or more, or 50% or more hepatocytes. .
  • the functions of cells, etc. include basic functions such as cell proliferation, repair, metabolism, and information exchange between cells, as well as functions according to the type of each cell.
  • Functions according to the type of each cell for example, in the case of hepatocytes, include sugar regulation, fat synthesis, protein metabolism, alcohol metabolism, ammonia metabolism, cholesterol metabolism, drug metabolism, bile secretion, and the like. . These functions can be measured by known methods, and the measured values can be used as values indicating the functions of cells and the like.
  • the function of hepatocytes is preferably evaluated using a metabolic activity value, and more preferably evaluated using a CYP1A2 metabolic activity value.
  • the value indicating the function of the cells, etc. is preferably 55% or more, more preferably 60% or more, more preferably 60% or more, relative to the measured value 24 hours after seeding the cells, etc. When it is 65% or more, it can be said that the functions of cells and the like are normally maintained.
  • culturing is used in a broad sense including not only the growth and maintenance of cells and the like, but also processes such as seeding, passage, induction of differentiation, and induction of self-organization of cells and the like.
  • the medium and the like used for culture are not limited, and a medium suitable for the characteristics of the cells may be selected.
  • the culture of cells, etc. may be two-dimensional (including cases where cells spontaneously stratify) or three-dimensional.
  • the culture vessel which is one aspect of the present invention, can be cultured in a suitable oxygen environment according to the state of cells, etc. by changing the amount of oxygen supply during culture. Even in a three-dimensional culture in which cells are stacked on top of each other, cells proliferate and differentiate, and a high level of self-organization of cells is likely to occur.
  • Three-dimensional culture is the intentional culturing of cells three-dimensionally, and there are two types: the scaffold type, in which cells are cultured in scaffolds, and the scaffold-free type, in which cells are cultured in suspension as spheroids. Either type is acceptable, but the scaffold type is preferred.
  • the scaffold type Matrigel TM , collagen gel, laminin, alginate hydrogel, and vitrigel are preferable scaffold materials because cells can be efficiently cultured.
  • the medium used for culture is not limited, it is preferable to culture the cells in the presence of serum (eg, fetal bovine serum) in order to culture the cells efficiently.
  • serum eg, fetal bovine serum
  • the cell seeding density for culturing using the culture vessel of one embodiment of the present invention is not particularly limited as long as the cells can proliferate and differentiate, but is preferably 0.1 ⁇ 10 5 cells/cm 2 to 10.1 ⁇ 10 5 cells/cm 2 . 0 ⁇ 10 5 cells/cm 2 , more preferably 0.3 ⁇ 10 5 cells/cm 2 to 5.0 ⁇ 10 5 cells/cm 2 , still more preferably 0.5 ⁇ 10 5 cells/cm 2 to 3.0 ⁇ 10 5 cells/cm 2 .
  • the cell seeding density is within the above range, the cells are more likely to adhere to the container and maintain normal cell functions than when the cell seeding density is outside the above range.
  • culture vessel means all vessels used for culturing cells, tissues or organs in culture medium. Cultivating in a medium means that at least a part of the cells or the like is immersed in the medium and cultured, and the entire cells or the like may not be immersed in the medium.
  • the culture vessel means all vessels used for culturing cells, tissues or organs. The culturing may be performed in medium.
  • Culture vessels include, for example, dishes, flasks, plates, bottles, bags, tubes and the like.
  • the culture vessels are typically used in devices such as incubators, mass culture devices, or perfusion culture devices.
  • the culture vessel is preferably a dish, flask or plate, more preferably a plate with at least one well.
  • the culture vessel is preferably a culture vessel having at least one well, more preferably a plate having at least one well, 6 wells, 12 wells, 24 wells, 48 wells, 96 wells, 384 wells, More preferred are plates with multiple wells, such as 1536 wells.
  • a culture vessel having a concave bottom surface such as a well needs to have a thick bottom surface in order to stabilize the complex shape of the bottom surface, which makes it difficult to sufficiently supply oxygen to cells and the like.
  • a plate having a plurality of wells such as 96 wells, 384 wells, 1536 wells, etc. has a stable shape, and the amount of oxygen supplied to cells and the like can be easily adjusted.
  • At least part of the bottom member has at least an oxygen permeable layer (Ib) and a gas barrier layer (II-b), and the gas barrier layer (II-b) is the Even if it is superimposed under the oxygen-permeable layer (Ib), similarly, even if it is a plate having a plurality of wells, the shape is stable and the amount of oxygen supplied to cells etc. is easy to adjust. .
  • the culture vessel ( ⁇ ) has a bottom member and a side wall member, and at least one culture space is formed by joining the bottom member and the side wall member.
  • the bottom member constitutes the bottom surface of the culture vessel by being joined to the side wall member.
  • the culture space is a limited space for culturing cells and the like, in other words, a space in which cells and the like to be cultured can freely move.
  • the bottom member and the side wall member are joined together so that the cells or the medium in the culture space do not leak out.
  • the bonding may be mechanical bonding, material bonding, or adhesive bonding, but adhesive bonding is preferred because it facilitates the combination of the bottom member and side wall members made of desired materials.
  • the culture vessel ( ⁇ ) is a culture vessel having at least one well
  • the culture vessel has, for example, a bottom member and a well side wall member, and the bottom member and the well side wall member are joined. may be formed with at least one well.
  • the bottom member constitutes the bottom of the well by joining with the well side wall member.
  • the culture vessel is preferably a culture vessel whose bottom surface includes a culture surface in order to hold or store a culture medium.
  • the culture surface means, of the parts constituting the culture vessel, the part with which the medium and/or the cells, etc. are in contact when the cells, etc. are cultured, or the part that the medium and/or the cells, etc. are expected to contact.
  • the bottom portion usually includes a culture surface, and the medium and/or cells contact the upper side of the culture surface, that is, the inside of the culture vessel.
  • the shape of the bottom surface of the culture vessel is not particularly limited, and includes flat bottom (F bottom), round bottom (U bottom), conical bottom (V bottom), flat bottom + curved edge, and the like.
  • a round bottom (U bottom), flat bottom (F bottom), conical bottom (V bottom), flat bottom + curved edge, etc. it may be processed at once by general injection molding or press molding, or a film Alternatively, it is also possible to prepare a sheet and perform secondary processing such as vacuum forming or pressure forming.
  • the shape of the bottom surface is selected according to the purpose of culture. For two-dimensional culture of cells, etc., a flat bottom (F bottom) is usually desirable, and for three-dimensional culture, a round bottom (U bottom) is desirable. bottom) or a conical bottom (V-bottom) is usually desirable.
  • the culture vessel may be coated with a natural polymeric material, a synthetic polymeric material, or an inorganic material at least on its culture surface, preferably on the side of the culture surface that comes into contact with the medium and/or cells. Coating can be performed by a known method.
  • a coated culture vessel is more excellent in adhesion and proliferation of cells and the like. This is probably because the natural polymer material, synthetic polymer material, or inorganic material coated on the culture surface serves as a scaffold for cells and the like. Therefore, when cells and the like are adhered and cultured, the culture surface of the culture vessel is preferably coated with a natural polymer material, a synthetic polymer material, or an inorganic material.
  • the side with which cells and the like come into contact is more preferably coated with a natural polymeric material, a synthetic polymeric material, or an inorganic material.
  • natural polymeric materials, synthetic polymeric materials, or inorganic materials are not particularly limited, but natural polymeric materials include collagen, gelatin, alginic acid, hyaluronic acid, glycosaminoglycans such as chondroitin sulfate, fibronectin, laminin, fibrinogen, Osteopontin, tenascin, vitronectin, thrombospondin, agarose, elastin, keratin, chitosan, fibrin, fibroin, sugars; synthetic polymer materials such as polyglycolic acid, polylactic acid, polyethylene glycol, polycaprolactone, synthetic peptides, synthetic proteins , polyhydroxyethyl methacrylate, polyethyleneimine; and inorganic materials such as ⁇ -tricalcium phosphate and calcium carbonate.
  • synthetic polymer materials such as polyglycolic acid, polylactic acid, polyethylene glycol, polycaprolactone, synthetic peptides, synthetic proteins , polyhydroxyeth
  • examples of the natural polymer material, synthetic polymer material, or inorganic material include vitrigel obtained by rehydrating after vitrifying a conventional hydrogel such as an extracellular matrix component.
  • a conventional hydrogel such as an extracellular matrix component.
  • collagen vitrigel composed of a high-density collagen fiber network made from collagen, which is one of the extracellular matrix components, can be used.
  • coating with proteins such as collagen, gelatin, laminin, polylysine, or peptides is preferable, and laminin, collagen or A coating treatment with polylysine is more preferred, and a coating treatment with collagen is even more preferred.
  • proteins such as collagen, gelatin, laminin, polylysine, or peptides
  • laminin, collagen or A coating treatment with polylysine is more preferred, and a coating treatment with collagen is even more preferred.
  • These coatings may be used singly or in combination of two or more.
  • the culture vessel may be microfabricated on the culture surface, preferably the side of the culture surface that comes into contact with the medium and/or cells, etc., in order to form spheroids and improve scaffolding functions for cells.
  • Microfabrication methods include cutting, optical lithography, electron beam direct writing, particle beam processing, scanning probe processing, self-organization of fine particles, and nanoimprinting from masters formed by these methods. It can be appropriately selected from a molding method represented by a molding method, a casting method, an injection molding method, a plating method, and the like.
  • the shape of the microfabrication is not particularly limited, it is preferable that the height from the groove portion to the peak portion is 20 nm to 500 ⁇ m. In addition, it is possible to reduce the thickness of the thinnest part of the culture vessel to 20 ⁇ m, compared to the case where the surface is not microfabricated.
  • a microfabricated culture vessel may be used as a microfluidic device (also called a microfluidic chip).
  • a microfluidic device is a general term for devices for applying microfabrication to the culture surface of a culture vessel to fabricate microfluidic channels and reaction vessels for bioresearch and chemical engineering.
  • micro TAS micro total analysis systems
  • Lab on a Chip which are being applied as next-generation culture apparatuses.
  • At least the culture surface of the culture vessel is preferably subjected to a surface modification treatment.
  • the method used for the surface modification treatment is not particularly limited, and examples include hydrophilic treatments such as corona treatment, plasma treatment, ozone treatment, and ultraviolet treatment; Vapor deposition, etching, addition of specific functional groups such as hydroxyl group, amino group, sulfone group, thiol group, carboxyl group, treatment with specific functional groups such as silane coupling, titanium coupling, zirconium coupling, oxidizing agent and physical treatments such as rubbing and sandblasting.
  • These surface modification treatments may be performed singly or in combination of two or more.
  • At least the culture surface of the culture vessel is preferably hydrophilized, more preferably corona-treated or plasma-treated.
  • hydrophilizing the surface of the culture vessel By hydrophilizing the surface of the culture vessel, the wettability of the surface of the culture vessel is increased, the adhesion between the culture vessel and cells is improved, and the cells can be uniformly grown on the surface of the culture vessel.
  • the natural polymer material, synthetic polymer material, or inorganic material is uniformly loaded on the culture surface of the culture vessel, and it becomes easier to adhere.
  • natural polymer materials, synthetic polymer materials, or inorganic materials do not peel off and can be used for cell culture while maintaining a stable initial state.
  • nitrogen, hydrogen, helium, oxygen, argon, or the like is used as the accompanying gas, and at least one gas selected from nitrogen, helium, and argon is preferably selected.
  • the culture vessel may be disinfected or sterilized to prevent contamination.
  • the method of disinfection or sterilization is not particularly limited, and physical disinfection methods such as the circulation steam method, boiling method, intermittent method, and ultraviolet method, chemical disinfection methods using gases such as ozone, and disinfectants such as ethanol; Heat sterilization methods such as high pressure steam method and dry heat method; irradiation sterilization methods such as gamma ray method, electron beam method and high frequency method; gas sterilization methods such as ethylene oxide gas method and hydrogen peroxide gas plasma method.
  • ethanol sterilization, autoclave sterilization, gamma ray sterilization, electron beam sterilization, or ethylene oxide gas sterilization is preferred because of its simple operation and sufficient sterilization.
  • oxygen-permeable layer (I) ⁇ Oxygen permeable layer (I)>
  • the oxygen-permeable layer (Ia) and the oxygen-permeable layer (Ib) described below are also collectively referred to as the oxygen-permeable layer (I).
  • the oxygen-permeable layer (Ia) is a layer that satisfies the following requirements (1) and (2).
  • the oxygen permeability P (Ia) at a temperature of 23°C and a humidity of 0% is 20,000 to 60,000 cm 3 /(m 2 ⁇ 24h ⁇ atm).
  • the measured total light transmittance is 70% or more
  • the oxygen permeability P(Ia) can be measured by the following method.
  • a measurement sample was prepared from the oxygen permeable layer (Ia), and the oxygen permeability coefficient [cm 3 ⁇ mm/(m 2 ⁇ 24 h ⁇ atm) at a temperature of 23 ° C. and a humidity of 0% was measured by a differential pressure type gas permeability measurement method. ] is measured.
  • the instrument used for the measurement is not particularly limited as long as it uses a differential pressure type gas permeability measurement method.
  • a measurement sample is preferably prepared by cutting a 50 ⁇ m-thick 90 ⁇ 90 mm test piece from the oxygen-permeable layer (Ia), and the diameter of the measurement portion is preferably 70 mm (permeation area is 38.46 cm 2 ). Since the oxygen permeability is high, it is more preferable to apply an aluminum mask to the sample in advance so that the actual permeation area is 5.0 cm 2 .
  • the sample to be measured may or may not have undergone microfabrication or surface modification treatment, but preferably has no treatment.
  • a value obtained by dividing the oxygen permeability coefficient by the film thickness ( ⁇ m) is defined as the oxygen permeability [cm 3 /(m 2 ⁇ 24h ⁇ atm)].
  • the oxygen permeability P (Ia) of the oxygen permeable layer (Ia) is preferably 20000 to 50000 cm 3 /(m 2 ⁇ 24h ⁇ atm), more preferably 25000 to 45000 cm 3 /(m 2 ⁇ 24h ⁇ atm), more preferably 30,000 to 40,000 cm 3 /(m 2 ⁇ 24h ⁇ atm).
  • the fact that the oxygen permeability P(Ia) is within the above range means that the oxygen permeable layer (Ia) is excellent in oxygen permeability.
  • the oxygen permeability P(Ia) of the oxygen-permeable layer (Ia) is lower than the above lower limit, the oxygen concentration in the medium will be low and cells will not proliferate and differentiate sufficiently.
  • the oxygen permeability P(Ia) is higher than the upper limit, the oxygen concentration in the medium becomes too high, and oxygen stress reduces cell function.
  • the oxygen permeability P(Ia) is within the above upper and lower limits, cells and the like can efficiently proliferate and differentiate according to the culture period, and maintain cell functions.
  • the oxygen permeability P (Ia) of the oxygen permeable layer (Ia) is adjusted, for example, by adjusting the thickness of the oxygen permeable layer (Ia) or the material constituting the oxygen permeable layer (Ia). can be adjusted by doing
  • the total light transmittance of the oxygen-permeable layer (Ia) is preferably 90.0% or higher, more preferably 92.0% or higher. Although there is no particular upper limit for the total light transmittance, it is usually 99.9%. It can be said that the fact that the total light transmittance is in the above range means that the oxygen-permeable layer (Ia) has excellent transparency.
  • the oxygen-permeable layer (Ia) has low transparency, making it difficult to observe cells and the like in the culture vessel.
  • the oxygen-permeable layer (Ia) has excellent transparency, so cells and the like can be easily observed with the naked eye or under a microscope.
  • the total light transmittance of the oxygen-permeable layer (Ia) can be adjusted, for example, by adjusting the thickness of the oxygen-permeable layer (Ia) or the material constituting the oxygen-permeable layer (Ia). can do.
  • the oxygen-permeable layer (Ib) is a layer that satisfies the following requirement (6).
  • the oxygen permeability P(Ib) can be measured by the same method and preferred mode as the oxygen permeability P(Ia).
  • the oxygen permeability P (Ib) of the oxygen permeable layer (Ib) is preferably 20,000 to 60,000 cm 3 /(m 2 ⁇ 24h ⁇ atm), more preferably 20,000 to 50,000 cm 3 /(m 2 ⁇ 24h ⁇ atm), more preferably 25,000 to 45,000 cm 3 /(m 2 ⁇ 24 h ⁇ atm), particularly preferably 30,000 to 40,000 cm 3 /(m 2 ⁇ 24 h ⁇ atm).
  • the fact that the oxygen permeability P(Ib) is within the above range means that the oxygen permeable layer (Ib) is excellent in oxygen permeability.
  • the oxygen permeability P (Ib) of the oxygen permeable layer (Ib) is less than 6.0 times the oxygen permeability P (II-b) of the gas barrier layer (II-b) described later, The oxygen concentration in the body becomes low, and cells do not grow and differentiate sufficiently.
  • the oxygen permeability P(Ib) is 60,000 cm 3 /(m 2 ⁇ 24 h ⁇ atm) or less, the oxygen concentration in the medium can be prevented from becoming too high, and the cell function due to oxygen stress can be prevented. It is easy to suppress the decline.
  • the oxygen permeability P(Ib) is within the above upper and lower limits, cells and the like can efficiently proliferate and differentiate according to the culture period, and maintain cell functions.
  • the oxygen permeability P (Ib) of the oxygen permeable layer (Ib) is adjusted, for example, by adjusting the thickness of the oxygen permeable layer (Ib) or the material constituting the oxygen permeable layer (Ib). can be adjusted by doing
  • Total light transmittance of oxygen-permeable layer (Ib) The total light transmittance of the oxygen permeable layer (Ib) can be measured by the same method as for the oxygen permeable layer (Ia).
  • the total light transmittance of the oxygen-permeable layer (Ib) is preferably 70% or higher, more preferably 90.0% or higher, still more preferably 92.0% or higher. Although there is no particular upper limit for the total light transmittance, it is usually 99.9%. It can be said that the fact that the total light transmittance is in the above range means that the oxygen-permeable layer (Ib) has excellent transparency.
  • the oxygen-permeable layer (Ib) will have low transparency, making it difficult to observe cells in the culture vessel.
  • the oxygen-permeable layer (Ib) has excellent transparency, so cells and the like can be easily observed with the naked eye or under a microscope.
  • the total light transmittance of the oxygen-permeable layer (Ib) can be adjusted, for example, by adjusting the thickness of the oxygen-permeable layer (Ib) or the material constituting the oxygen-permeable layer (Ib). can do.
  • the upper surface of the oxygen permeable layer (I) is usually in contact with the culture medium and/or cells.
  • the water contact angle of the oxygen permeable layer (I) is preferably 30° or more, more preferably 35° or more, still more preferably 40° or more, particularly preferably 45° or more, and preferably 120° or less, more preferably 110° or less, more preferably 100° or less, particularly preferably 84° or less, and most preferably 70° or less.
  • the oxygen-permeable layer (I) By adjusting the water contact angle of the oxygen-permeable layer (I) within the above range, for example, it becomes easier for cells and the like to adhere to the oxygen-permeable layer (I), and the cells and the like grow uniformly on the oxygen-permeable layer (I). Cheap.
  • the oxygen-permeable layer (I) is uniformly coated with a natural polymer material, a synthetic polymer material, or an inorganic material, making it easier to adhere. In this environment, natural polymer materials, synthetic polymer materials, or inorganic materials do not peel off and can be used for cell culture while maintaining a stable initial state.
  • the method for measuring the water contact angle is not particularly limited, and a known method can be used, preferably the sessile drop method.
  • the shape of the water droplet can be regarded as spherical under constant temperature and humidity conditions of 25 ⁇ 5 ° C. and 50 ⁇ 10% according to Japanese Industrial Standard JIS-R3257 (test method for wettability of substrate glass surface).
  • a water droplet with a volume of 4 ⁇ L or less is dropped on the surface of the measurement sample, and the angle of the contact interface between the measurement sample and the water droplet is measured within 1 minute after the water droplet contacts the measurement sample surface by the sessile drop method. can do.
  • the water contact angle of the oxygen permeable layer (I) can be adjusted, for example, by adjusting the material constituting the oxygen permeable layer (I), the surface modification treatment conditions of the oxygen permeable layer (I), and the like.
  • the material constituting the oxygen-permeable layer (Ia) is not particularly limited as long as it can form the oxygen-permeable layer (Ia) satisfying the requirements (1) and (2).
  • the material constituting the oxygen-permeable layer (Ib) is not particularly limited as long as it can form the oxygen-permeable layer (Ib) satisfying the requirement (6).
  • the materials constituting the oxygen-permeable layer (I) may be used singly or in combination of two or more.
  • An appropriate material can be selected for the material constituting the oxygen-permeable layer (I) from the viewpoints of moldability, transparency, shape stability, lightness, low drug sorption, autofluorescence, radiation resistance, and the like.
  • the culture vessel of one aspect of the present invention can efficiently culture cells and the like, and the morphology of the cells and the like can be improved. It is easier to observe and easier to use for drug discovery screening applications and diagnostic applications.
  • fluorine-based resins include polytetrafluoroethylene (PTFE), polytrifluorochloroethylene, polyvinyl fluoride, polyvinylidene fluoride, dichlorodifluoroethylene, polychlorotrifluoroethylene, fluorinated ethylene propylene copolymer, and perfluoroalkylvinyl ether. polymers, perfluoroalkyl vinyl ester polymers, ethylenetetrafluoroethylene copolymers, polydimethylsiloxane (PDMS), and the like.
  • PTFE polytetrafluoroethylene
  • PDMS polydimethylsiloxane
  • polyolefin resins that can be used as the oxygen permeable layer (I) include homopolymers of ⁇ -olefins such as ethylene, 1-butene, 1-hexene, 4-methyl-1-pentene, and 1-octene, or Copolymer; high-pressure low-density polyethylene; linear low-density polyethylene (LLDPE); high-density polyethylene; polypropylene (propylene homopolymer); copolymerization of propylene and ⁇ -olefin having 2 to 10 carbon atoms coalescence; ethylene/vinyl acetate copolymer (EVA); ionomer resin; fluorine-containing cyclic olefin polymer and the like.
  • ⁇ -olefins such as ethylene, 1-butene, 1-hexene, 4-methyl-1-pentene, and 1-octene, or Copolymer
  • high-pressure low-density polyethylene linear low-density polyethylene
  • Polyolefin resins are preferable from the viewpoint of excellent balance of moldability, transparency, shape stability, lightness, low drug sorption, autofluorescence, etc.
  • 4-methyl-1-pentene polymer described later (X) is preferred. That is, the oxygen permeable layer (I) preferably contains the 4-methyl-1-pentene polymer (X).
  • Copolymers of 4-methyl-1-pentene which is an example of the 4-methyl-1-pentene polymer (X), with other monomers include random copolymers, alternating copolymers, and block copolymers. Either a coalescence or a graft copolymer may be used.
  • Copolymers of 4-methyl-1-pentene with other monomers include 4-methyl-1-pentene, ethylene and ⁇ -olefins having 3 to 20 carbon atoms (excluding 4-methyl-1-pentene A copolymer with at least one olefin selected from ) is preferred because it has high strength, is resistant to tearing and cracking even when used as the oxygen-permeable layer (I), and has little deflection.
  • the 4-methyl-1-pentene polymer (X) includes 4-methyl-1-pentene homopolymer and 4-methyl-1-pentene, ethylene and an ⁇ -olefin having 3 to 20 carbon atoms (4- It is preferably at least one polymer selected from copolymers with at least one olefin selected from (excluding methyl-1-pentene), 4-methyl-1-pentene, ethylene and carbon number More preferably, it is a copolymer with at least one olefin selected from 3 to 20 ⁇ -olefins (excluding 4-methyl-1-pentene).
  • olefin examples include ethylene, propylene, 1-butene, 1-hexene, 1-heptene, 1-octene, 1-decene, 1-tetradecene, 1-hexadecene, 1-heptadecene, 1-octadecene, and 1-eicosene. are mentioned.
  • the olefin can be appropriately selected according to physical properties required for the oxygen-permeable layer (I).
  • the olefins are preferably ⁇ -olefins having 8 to 18 carbon atoms from the viewpoint of appropriate oxygen permeability and excellent rigidity, such as 1-octene, 1-decene, 1-dodecene, 1-tetradecene, At least one selected from 1-hexadecene, 1-heptadecene and 1-octadecene is more preferred.
  • the number of carbon atoms in the olefin is within the above range, the processability of the polymer is improved, and the appearance of the oxygen permeable layer (I) due to cracks and cracks at the edges tends to be less likely to occur. In addition, the defective rate of the oxygen-permeable layer (I) is reduced.
  • the olefin can be used alone or in combination of two or more.
  • the number of carbon atoms is preferably 2 or more, more preferably 10 or more.
  • the content of structural units derived from 4-methyl-1-pentene in the 4-methyl-1-pentene polymer (X) is preferably 60 to 100 mol%, more preferably 80 to 99.5 mol%, More preferably 85 to 98 mol %. Further, the 4-methyl-1-pentene polymer (X) is selected from 4-methyl-1-pentene, ethylene and ⁇ -olefins having 3 to 20 carbon atoms (excluding 4-methyl-1-pentene).
  • a copolymer with at least one olefin at least one olefin selected from ethylene and ⁇ -olefins having 3 to 20 carbon atoms (excluding 4-methyl-1-pentene) in the copolymer
  • the content of the structural unit derived from is preferably 0 to 40 mol%, more preferably 0.5 to 20 mol%, still more preferably 2 to 15 mol%.
  • the content of these structural units is based on 100 mol % of the total amount of repeating structural units in the 4-methyl-1-pentene polymer (X).
  • the 4-methyl-1-pentene polymer (X) is a structural unit derived from 4-methyl-1-pentene and the ethylene and an ⁇ -olefin having 3 to 20 carbon atoms, as long as the effects of the present invention are not impaired. It may have structural units other than structural units derived from (hereinafter also referred to as "other structural units"). The content of other structural units is, for example, 0 to 10.0 mol %. When the 4-methyl-1-pentene polymer (X) has other structural units, the other structural units may be one or more.
  • Examples of monomers that lead to other structural units include cyclic olefins, aromatic vinyl compounds, conjugated dienes, non-conjugated polyenes, functional vinyl compounds, hydroxyl group-containing olefins, and halogenated olefins.
  • Cyclic olefins, aromatic vinyl compounds, conjugated dienes, non-conjugated polyenes, functional vinyl compounds, hydroxyl group-containing olefins and halogenated olefins include, for example, paragraphs [0035] to [0041] of JP-A-2013-169685. compounds can be used.
  • the 4-methyl-1-pentene polymer (X) may be used singly or in combination of two or more.
  • a commercial product can also be used as the 4-methyl-1-pentene polymer (X).
  • TPX MX001, MX002, MX004, MX0020, MX021, MX321, RT18, RT31 or DX845 (all trademarks) manufactured by Mitsui Chemicals, Inc. may be used.
  • 4-methyl-1-pentene polymer (X) manufactured by other manufacturers can be preferably used as long as it satisfies the above requirements.
  • the 4-methyl-1-pentene polymer (X) usually has a melting point of 200°C to 240°C and high heat resistance. In addition, since it does not cause hydrolysis and has excellent water resistance, boiling water resistance, and steam resistance, the oxygen permeable layer (I) containing the 4-methyl-1-pentene polymer (X) can be sterilized with high pressure steam. is.
  • the 4-methyl-1-pentene polymer (X) has a high visible light transmittance (usually 90% or more) and does not emit autofluorescence, so the 4-methyl-1-pentene polymer (X) is
  • the culture vessel formed from the oxygen-permeable layer (I) containing the oxygen-permeable layer (I) facilitates observation of cultured cells.
  • the 4-methyl-1-pentene polymer (X) has high strength, the culture vessel formed from the oxygen-permeable layer (I) containing the 4-methyl-1-pentene polymer (X) is hard to break, Less deflection.
  • the 4-methyl-1-pentene polymer (X) can be heat-sealed, and can be easily heat-sealed not only with itself but also with other materials.
  • it can be thermoformed, it can be easily formed into a culture vessel of any shape, for example, it can be easily formed using an imprint method or an insert method.
  • the weight average molecular weight (Mw) of the 4-methyl-1-pentene polymer (X), measured by gel permeation chromatography (GPC) using standard polystyrene as a reference material, is preferably 10,000 to 2,000,000, or more. It is preferably 20,000 to 1,000,000, more preferably 30,000 to 500,000.
  • the sample concentration for GPC measurement can be, for example, 1.0 to 5.0 mg/mL.
  • the molecular weight distribution (Mw/Mn) of the 4-methyl-1-pentene polymer (X) is preferably 1.0 to 30, more preferably 1.1 to 25, still more preferably 1.1 to 20. be.
  • the solvent used in GPC is preferably ortho-dichlorobenzene.
  • the conditions shown in Examples to be described later can be cited, but the measurement conditions are not limited to these.
  • the film produced by melt molding in the molding method of the 4-methyl-1-pentene polymer (X) described later suppresses the occurrence of defects such as gel. It is easy to do, and it becomes easy to form a film with a uniform surface.
  • the solubility in the solvent is improved, so that problems such as film gel can be easily suppressed, making it easier to form a film with a uniform surface.
  • the weight average molecular weight (Mw) to the above lower limit or more, the strength of the culture vessel tends to be sufficient. Furthermore, by setting the molecular weight distribution within the above range, it is easy to suppress the stickiness of the surface of the culture vessel produced, and the toughness of the culture vessel tends to be sufficient, so bending during molding and cracking during cutting are prevented. easier to suppress.
  • the weight-average molecular weight (Mw) and molecular weight distribution (Mw/Mn) of the 4-methyl-1-pentene polymer (X) were obtained by using two or more types of 4-methyl-1-pentene polymer (X). In that case, Mw and Mw/Mn should be within the above ranges.
  • the 4-methyl-1-pentene polymer (X) preferably has an oxygen permeability coefficient of 1000 to 3000 cm 3 ⁇ mm/(m 2 ⁇ 24h ⁇ atm), more preferably 1000 to 2500 cm 3 ⁇ mm/(m 2 ⁇ 24 h ⁇ atm) is more preferable.
  • the oxygen permeability coefficient is in the above range, the oxygen permeability is excellent, so that cells and the like maintain good morphology and are likely to proliferate efficiently according to the culture period.
  • the oxygen-permeable layer (I ) has no adverse effect on the culture, has good shape stability, transparency, moldability, and oxygen permeability, can be sterilized, and can be used for culturing cells. It is very excellent as a culture vessel used for
  • the method for producing the 4-methyl-1-pentene polymer (X) may be any method as long as it can polymerize 4-methyl-1-pentene, olefin and other monomers. Also, a chain transfer agent such as hydrogen may coexist in order to control the molecular weight and molecular weight distribution. Equipment used for manufacturing is also not limited.
  • the polymerization method may be a known method such as a gas phase method, a slurry method, a solution method, or a bulk method. A slurry method and a solution method are preferred.
  • the polymerization method may be a single-stage polymerization method or a multi-stage polymerization method such as a two-stage polymerization method, in which a plurality of polymers having different molecular weights are blended in a polymerization system.
  • hydrogen When hydrogen is used as a chain transfer agent in either a single-stage polymerization method or a multi-stage polymerization method, it may be charged all at once or dividedly, for example, at the beginning, middle and end of the polymerization.
  • the polymerization may be carried out at normal temperature, or may be heated if necessary, but from the viewpoint of polymerization efficiency, it is preferably carried out at 20°C to 80°C, more preferably at 40°C to 60°C. .
  • the catalyst used for production is also not limited, but from the viewpoint of polymerization efficiency, for example, the solid titanium catalyst component (I) described in WO 2006/054613 and the transition metal compound described in WO 2014/050817 are used. It is preferable to use an olefin polymerization catalyst (metallocene catalyst) containing (A).
  • the oxygen-permeable layer (I) containing the 4-methyl-1-pentene polymer (X) is made of a composition containing the 4-methyl-1-pentene polymer (X), the composition is 100 mass. %, the 4-methyl-1-pentene polymer (X) is preferably 90% by mass or more and less than 100% by mass, more preferably 95% by mass or more and less than 100% by mass, and particularly preferably 99% by mass. % or more and less than 100% by mass. If a large amount of components other than the 4-methyl-1-pentene polymer (X) is contained, not only oxygen permeability is lowered, but also transparency and strength are lowered.
  • Components other than the 4-methyl-1-pentene polymer (X) include a heat stabilizer, a light stabilizer, a processing aid, a plasticizer, an antioxidant, a lubricant, an antifoaming agent, an antiblocking agent, and a coloring agent. agents, modifiers, antibacterial agents, antifungal agents, antifogging agents, and other additives.
  • the oxygen-permeable layer (I) may have a single-layer structure containing only one layer or a multilayer structure containing two or more layers. Moreover, when the oxygen-permeable layer (I) has a multilayer structure, the constituent materials and thickness of each layer may be the same or different.
  • the thickness of the oxygen permeable layer (Ia) is not particularly limited as long as the oxygen permeability at a temperature of 23° C. and a humidity of 0% is 20,000 to 60,000 cm 3 /(m 2 ⁇ 24 h ⁇ atm), but it is preferable. is 20 to 500 ⁇ m, more preferably 25 to 500 ⁇ m, still more preferably 30 to 200 ⁇ m.
  • the thickness of the oxygen-permeable layer (Ib) is not particularly limited as long as it satisfies requirement (6), but is preferably 20-500 ⁇ m, more preferably 25-500 ⁇ m, still more preferably 30-200 ⁇ m.
  • the thickness means the total thickness of each layer when the oxygen-permeable layer (I) has a multi-layer structure.
  • the thickness of the oxygen-permeable layer (I) is within the above range, the strength is excellent. In particular, even when the number of wells in the culture vessel is small and the hole diameter is large, bending is less likely to occur. In addition, it is difficult to break when corona treatment or the like is applied.
  • gas barrier layer (II) ⁇ Gas barrier layer (II)>
  • gas barrier layer (II-a) and gas barrier layer (II-b) described below are also collectively referred to as gas barrier layer (II).
  • the gas barrier layer (II-a) is a layer that satisfies the following requirements (3) and (4).
  • (3) Oxygen permeability P(II-a) at a temperature of 23° C. and a humidity of 0% is 3000 cm 3 /(m 2 ⁇ 24 h ⁇ atm) or less.
  • the total light transmittance is 70% or more
  • the oxygen permeability P(II-a) can be measured by the same method as for the oxygen-permeable layer (Ia).
  • the oxygen permeability P(II-a) of the gas barrier layer (II-a) is preferably greater than 1 cm 3 /(m 2 ⁇ 24 h ⁇ atm) and 3000 cm 3 /(m 2 ⁇ 24 h ⁇ atm) or less, more preferably is greater than 1 cm 3 /(m 2 ⁇ 24 h ⁇ atm) and less than or equal to 1000 cm 3 /(m 2 ⁇ 24 h ⁇ atm), more preferably greater than 1 cm 3 /(m 2 ⁇ 24 h ⁇ atm) and 500 cm 3 /(m 2 ⁇ 24 h ⁇ atm) or less, particularly preferably more than 1 cm 3 /(m 2 ⁇ 24 h ⁇ atm) and 300 cm 3 /(m 2 ⁇ 24 h ⁇ atm) or less.
  • the fact that the oxygen permeability P(II-a) is within
  • the oxygen permeability P(II-a) of the gas barrier layer (II-a) is too high, the oxygen concentration in the medium immediately after seeding the cells becomes too high, resulting in poor adhesion of the cells. If the oxygen permeability P(II-a) of the gas barrier layer (II-a) is too low, the adhesiveness of cells, etc. will also deteriorate, and the cell function will tend not to be maintained in subsequent cultures. When the oxygen permeability P(II-a) is within the above upper and lower limits, cells and the like adhere well to the culture vessel.
  • the oxygen permeability P(II-a) of the gas barrier layer (II-a) can be determined, for example, by adjusting the thickness of the gas barrier layer (II-a), the material constituting the gas barrier layer (II-a), or the like. , can be adjusted.
  • the total light transmittance can be measured by the same method as for the oxygen-permeable layer (Ia).
  • the total light transmittance of the gas barrier layer (II-a) is preferably 85.0% or higher, more preferably 90.0% or higher. Although there is no particular upper limit for the total light transmittance, it is usually 99.9%. It can be said that the fact that the total light transmittance is within the above range means that the gas barrier layer (II-a) has excellent transparency.
  • the total light transmittance is too low, the transparency of the gas barrier layer (II-a) is low, making it difficult to observe cells and the like in the culture vessel.
  • the gas barrier layer (II) has excellent transparency, so cells and the like can be easily observed with the naked eye or under a microscope.
  • the total light transmittance of the gas barrier layer (II-a) can be adjusted, for example, by adjusting the thickness of the gas barrier layer (II-a) or the material constituting the gas barrier layer (II-a). can.
  • the gas barrier layer (II-b) is a layer that satisfies requirement (5) below.
  • Oxygen permeability P(II-b) at a temperature of 23° C. and a humidity of 0% is 3000 cm 3 /(m 2 ⁇ 24 h ⁇ atm) or less
  • the oxygen permeability P(II-b) can be measured by the same method and preferred mode as the oxygen permeability P(Ia).
  • a suitable range and adjusting method of the oxygen permeability P(II-b) of the gas barrier layer (II-b) are the same as those of the gas barrier layer (II-a).
  • the total light transmittance of the gas barrier layer (II-b) can be measured by the same method as for the oxygen permeable layer (Ia).
  • the total light transmittance of the gas barrier layer (II-b) is preferably 70% or higher, more preferably 85.0% or higher, and even more preferably 90.0% or higher. Although there is no particular upper limit for the total light transmittance, it is usually 99.9%. It can be said that the fact that the total light transmittance is within the above range means that the gas barrier layer (II-b) has excellent transparency.
  • the total light transmittance is too low, the transparency of the gas barrier layer (II-b) is low, making it difficult to observe cells and the like in the culture vessel.
  • the gas barrier layer (II-b) has excellent transparency, so that cells and the like can be easily observed with the naked eye or under a microscope.
  • the total light transmittance of the gas barrier layer (II-b) can be adjusted, for example, by adjusting the thickness of the gas barrier layer (II-b) or the material constituting the gas barrier layer (II-b). can.
  • the material constituting the gas barrier layer (II-a) is not particularly limited as long as it can form the gas barrier layer (II-a) that satisfies requirements (3) and (4).
  • Examples of materials constituting the gas barrier layer (II-a) include polyethylene terephthalate (PET), nylon, ethylene-vinyl alcohol copolymer (EVOH), polyglycolic acid, aromatic polyamide, and polyvinylidene chloride (PVDC).
  • Appropriate materials can be selected from among these from the viewpoint of moldability, transparency, shape stability, lightness, low drug sorption, autofluorescence, radiation resistance, and the like. From this point of view, by selecting polyethylene terephthalate, the culture vessel which is one embodiment of the present invention can efficiently culture cells and the like, can easily observe the morphology of cells and the like, and can be used for drug discovery screening and diagnostic purposes. becomes easier to use. That is, the gas barrier layer (II-a) preferably contains polyethylene terephthalate (PET).
  • PET polyethylene terephthalate
  • the materials constituting the gas barrier layer (II-a) may be used singly or in combination of two or more.
  • the material constituting the gas barrier layer (II-b) is not particularly limited as long as it can form the gas barrier layer (II-b) satisfying the requirement (5).
  • Materials constituting the gas barrier layer (II-b) include, for example, polyethylene terephthalate (PET), nylon, ethylene-vinyl alcohol copolymer (EVOH), polyglycolic acid, aromatic polyamide, polyvinylidene chloride (PVDC), and polyolefin. Appropriate materials can be selected from among these from the viewpoint of moldability, transparency, shape stability, lightness, low drug sorption, autofluorescence, radiation resistance, and the like.
  • the culture vessel of one embodiment of the present invention can efficiently culture cells and the like, can easily observe the morphology of cells and the like, and can be used for drug discovery screening. Easier to use for diagnostic purposes. That is, the gas barrier layer (II-b) preferably contains at least one selected from the group consisting of polyethylene terephthalate (PET) and polyolefin, more preferably polyethylene terephthalate (PET). The materials constituting the gas barrier layer (II-b) may be used singly or in combination of two or more.
  • the gas barrier layer (II-a) may be an unstretched film or a stretched film such as a uniaxially stretched film or a biaxially stretched film. More specifically, such films include monolayer or multilayer oriented films such as biaxially oriented polyethylene terephthalate films and biaxially oriented nylon films, ethylene-vinyl alcohol copolymer (EVOH), polyglycolic acid, aromatic Examples include films made of polyamide, polyvinylidene chloride (PVDC), etc., and various polyvinylidene chloride-coated films.
  • PVDC polyvinylidene chloride
  • the gas barrier layer (II-b) may be an unstretched film or a stretched film such as a uniaxially stretched film or a biaxially stretched film. More specifically, such films include monolayer or multilayer oriented films such as biaxially oriented polyethylene terephthalate films and biaxially oriented nylon films, ethylene-vinyl alcohol copolymer (EVOH), polyglycolic acid, aromatic Examples include films made of polyamide, polyvinylidene chloride (PVDC), etc., various polyvinylidene chloride-coated films, and polyolefin films containing polyethylene (PE), polypropylene (PP), and the like.
  • PVDC polyvinylidene chloride
  • PE polypropylene
  • Polyethylene terephthalate is a polymer obtained by polycondensing at least one selected from terephthalic acid and its ester-forming derivatives with at least one selected from ethylene glycol and its ester-forming derivatives.
  • ester-forming derivatives of terephthalic acid include alkyl esters of terephthalic acid such as dimethyl terephthalate.
  • ester-forming derivatives of ethylene glycol include fatty acid esters such as ethylene glycol fatty acid esters.
  • Polyethylene terephthalate contains at least one selected from terephthalic acid and ester-forming derivatives thereof and at least one selected from other dicarboxylic acids and ester-forming derivatives thereof, provided that the effects obtained in the present invention are not inhibited. It may be a polymerized one, or a copolymer of at least one selected from ethylene glycol and its ester-forming derivatives and at least one selected from other diols and their ester-forming derivatives. good.
  • Dicarboxylic acids and ester-forming derivatives thereof used as copolymerization components include, for example, isophthalic acid, adipic acid, oxalic acid, sebacic acid, decanedicarboxylic acid, naphthalenedicarboxylic acid, and alkyl esters thereof.
  • Examples of diols and ester-forming derivatives thereof used as copolymer components include ethylene glycol, propylene glycol, neopentyl glycol, 1,5-pentanediol, 1,6-hexanediol, decamethylene glycol, cyclohexanediol, Examples include methanol, cyclohexanediol, long-chain glycols such as polyethylene glycol having a molecular weight of 400 to 6000, poly-1,3-propylene glycol and polytetramethylene glycol, and fatty acid esters thereof. These copolymer components may be used singly or in combination of two or more. These copolymer components are preferably 20% by mass or less of the raw material forming polyethylene terephthalate.
  • the type of polyethylene terephthalate is not particularly limited as long as it does not impair the effects obtained in the present invention, but from the viewpoint of mechanical strength and moldability, crystalline polyethylene terephthalate (C-PET) is preferred.
  • C-PET crystalline polyethylene terephthalate
  • a method for producing polyethylene terephthalate is not particularly limited, and a known method can be used.
  • polyolefins examples include ⁇ -olefins such as ethylene, 1-butene, 1-hexene, 4-methyl-1-pentene, and 1-octene.
  • Polymer or copolymer high pressure low density polyethylene; linear low density polyethylene (LLDPE); high density polyethylene; polypropylene (propylene homopolymer); propylene and ⁇ -olefin having 2 to 10 carbon atoms ethylene/vinyl acetate copolymer (EVA); ionomer resin; fluorine-containing cyclic olefin polymer, and the like.
  • the gas barrier layer (II-b) preferably contains a propylene homopolymer or copolymer, more preferably polypropylene (propylene homopolymer).
  • Structural units other than propylene-derived structural units in the propylene copolymer are not particularly limited, but may be at least one olefin selected from ethylene and ⁇ -olefins having 4 to 20 carbon atoms.
  • olefin examples include ethylene, 1-butene, 1-hexene, 1-heptene, 1-octene, 1-decene, 1-tetradecene, 1-hexadecene, 1-heptadecene, 1-octadecene, and 1-eicosene. be done.
  • the olefin can be appropriately selected depending on the physical properties required for the gas barrier layer (II-b).
  • film-like polyethylene terephthalate and/or polyolefin When film-like polyethylene terephthalate and/or polyolefin is used as the gas barrier layer (II), the film-like polyethylene terephthalate and/or polyolefin may or may not be stretched. Film-like polyethylene terephthalate and/or polyolefin are preferred from the viewpoint of mechanical properties because they are crystallographically oriented when stretched.
  • the stretching method is preferably biaxial stretching.
  • polyethylene terephthalate and polyolefin are preferably contained in 100% by mass of the composition. It is 90% by mass or more and less than 100% by mass, more preferably 95% by mass or more and less than 100% by mass, and particularly preferably 99% by mass or more and less than 100% by mass. If a large amount of components other than polyethylene terephthalate and polyolefin is contained, not only the oxygen permeability is increased, but also the transparency and strength are lowered.
  • Ingredients other than polyethylene terephthalate and polyolefin include heat stabilizers, light stabilizers, processing aids, plasticizers, antioxidants, lubricants, antifoaming agents, antiblocking agents, coloring agents, modifiers, and antibacterial agents. , anti-mold agents, anti-fogging agents, adhesives and other additives.
  • the gas barrier layer (II) may have a single layer structure containing only one layer or a multilayer structure containing two or more layers. Moreover, when the gas barrier layer (II) has a multilayer structure, the constituent materials and thickness of each layer may be the same or different.
  • one of them may be a deposited film layer composed of an inorganic oxide. This can further improve the gas barrier properties of the gas barrier layer (II).
  • inorganic oxides include aluminum oxide (alumina), silicon oxide (silica), magnesium oxide, calcium oxide, zirconium oxide, titanium oxide, boron oxide, hafnium oxide, barium oxide, and silicon oxide carbide (carbon-containing silicon oxide). etc. can be mentioned.
  • the thickness of the gas barrier layer (II) is not particularly limited as long as the oxygen permeability at a temperature of 23° C. and a humidity of 0% is 3000 cm 3 /(m 2 ⁇ 24 h ⁇ atm) or less, but is preferably 20 to 500 ⁇ m. , more preferably 25 to 500 ⁇ m, still more preferably 30 to 200 ⁇ m.
  • the thickness refers to the total thickness of each layer when the gas barrier layer (II) has a multilayer structure.
  • the thickness of the gas barrier layer (II) is within the above range, the strength is excellent. In particular, even when the number of wells in the culture vessel is small and the hole diameter is large, bending is less likely to occur. In addition, when the gas barrier layer (II) is attached and detached from the oxygen permeable layer (I), it is easy to handle and does not tear easily.
  • the gas barrier layer (II-b) preferably contains at least one selected from the group consisting of polyethylene terephthalate (PET) and polyolefin and satisfies the following requirements (7) or (8). (7) a mixture of at least one selected from the group consisting of polyethylene terephthalate (PET) and polyolefin, and (8) at least one selected from the group consisting of polyethylene terephthalate (PET) and polyolefin and a layer containing an adhesive
  • [Requirement (7)] Containing a mixture of at least one selected from the group consisting of polyethylene terephthalate (PET) and polyolefin and an adhesive means that one layer of the gas barrier layer (II-b) consists of polyethylene terephthalate (PET) and polyolefin It means that it is composed of a composition of at least one selected from the group and an adhesive.
  • the content of at least one selected from the group consisting of polyethylene terephthalate (PET) and polyolefin in the composition is not particularly limited.
  • the content of the adhesive in the composition is not particularly limited.
  • the mass ratio of at least one selected from the group consisting of polyethylene terephthalate (PET) and polyolefin to the adhesive is not particularly limited. The adhesive will be described later.
  • Having a layer containing at least one selected from the group consisting of polyethylene terephthalate (PET) and polyolefin and a layer containing an adhesive means that the gas barrier layer (II-b) comprises polyethylene terephthalate (PET) and polyolefin It means having at least two layers, a layer containing at least one selected from the group consisting of and a layer containing an adhesive.
  • the thickness of the layer containing at least one selected from the group consisting of polyethylene terephthalate (PET) and polyolefin is not particularly limited, but is preferably 10-500 ⁇ m, more preferably 50-100 ⁇ m.
  • the thickness of the layer containing the adhesive is not particularly limited, but is preferably 1 to 100 ⁇ m, more preferably 5 to 20 ⁇ m.
  • the thickness ratio of the layer containing at least one selected from the group consisting of polyethylene terephthalate (PET) and polyolefin and the layer containing the adhesive is not particularly limited, but is preferably 500:1 to 10:100, more preferably is 100:5 to 50:20. The adhesive will be described later.
  • At least part of the culture surface of the culture vessel ( ⁇ ) is an oxygen permeable layer (Ia) that satisfies requirements (1) and (2) and a gas barrier layer that satisfies requirements (3) and (4). It has a layered region with (II-a).
  • the culture vessel ( ⁇ ) Since the culture vessel ( ⁇ ) has a gas barrier layer (II-a), oxygen permeation into the interior of the vessel is suppressed.
  • the oxygen permeable layer (Ia) When the oxygen permeable layer (Ia) is exposed by removing the gas barrier layer (II-a) from the oxygen permeable layer (Ia), the culture vessel ( ⁇ ) allows sufficient oxygen permeation into the interior of the vessel. becomes.
  • FIG. 1 shows a conceptual diagram of the cross section of the culture vessel, which is one aspect of the present invention.
  • the left side of FIG. 1 shows the state of having the gas barrier layer (II) on the culture surface, and the right side of FIG. 1 shows the state of removing the gas barrier layer (II) from the oxygen permeable layer (I).
  • the culture surface has a laminated area of the oxygen permeable layer (Ia) and the gas barrier layer (II-a)
  • at least a part of the culture surface has Means a laminated region of the oxygen permeable layer (Ia) and the gas barrier layer (II-a), and the entire region of the culture surface is the oxygen permeable layer (Ia) and the gas barrier layer (II-a). or only a partial region of the culture surface may be a laminated region of the oxygen permeable layer (Ia) and the gas barrier layer (II-a).
  • the area of the culture surface other than the laminated area is the oxygen permeable layer (I- It is preferable to have a region composed of a), and all regions of the culture surface other than the laminated region are more preferably regions composed of the oxygen-permeable layer (Ia).
  • the bottom When the culture vessel ( ⁇ ) is, for example, a dish, flask or plate, the bottom usually includes the culture surface. -a) and the gas barrier layer (II-a). When at least part or all of the bottom surface has a laminated region of the oxygen permeable layer (Ia) and the gas barrier layer (II-a), it is easy to regulate oxygen supplied to the culture medium via the laminated region.
  • the culture vessel ( ⁇ ) preferably has a laminated region of the oxygen permeable layer (Ia) and the gas barrier layer (II-a) over the entire culture surface. That is, when the culture vessel ( ⁇ ) is, for example, a dish, flask or plate, the bottom surface includes the culture surface. It preferably has a laminated region with layer (II-a).
  • the ratio of the laminated area of the oxygen permeable layer (Ia) and the gas barrier layer (II-a) to the area of the culture surface is not particularly limited, but is preferably 80% or more, more preferably 90% or more, and still more preferably. is 95% or more.
  • the ratio of the laminated area of the oxygen permeable layer (Ia) and the gas barrier layer (II-a) to the area of the culture surface is within the above range, the amount of oxygen supplied to cells and the like can be easily adjusted.
  • the gas barrier layer (II-a) is provided on the lower surface of the oxygen permeable layer (Ia). That is, in the laminated region of the oxygen permeable layer (Ia) and the gas barrier layer (II-a), the oxygen permeable layer (Ia) and the gas barrier layer (II-a) are arranged from the inner side of the culture vessel ( ⁇ ). are stacked in this order.
  • the region of the oxygen permeable layer (Ia) and the region of the gas barrier layer (II-a) may have the same size, or one of them may be larger. Further, the area of the gas barrier layer (II-a) is preferably equal to or larger than the area of the oxygen permeable layer (Ia), since the gas barrier layer (II-a) can be easily attached and detached.
  • the area of the gas barrier layer (II-a) is preferably larger than the area of the culture surface because it is easy to adjust the oxygen supply amount and to attach and detach the gas barrier layer (II-a). ) is provided on the entire bottom surface of the culture vessel.
  • the area of the gas barrier layer (II-a) is preferably larger than the total bottom area of each well, and the gas barrier layer (II-a) is the entire bottom surface of the plate. It is preferable to provide
  • Materials constituting members other than the culture surface of the culture vessel ( ⁇ ) are not particularly limited, and known materials can be used. Such materials include, for example, polystyrene (PS), polydimethylsiloxane (PDMS), thermosetting resins, cyclic olefin polymers, cyclic olefin copolymers, glass, and the like.
  • Materials constituting members other than the laminated region of the oxygen permeable layer (Ia) and the gas barrier layer (II-a) on the culture surface of the culture vessel ( ⁇ ) are not particularly limited, and known materials can be used. can.
  • Such materials include, for example, polystyrene (PS), polydimethylsiloxane (PDMS), thermosetting resins, cyclic olefin polymers, cyclic olefin copolymers, glass, and the like.
  • PS polystyrene
  • PDMS polydimethylsiloxane
  • thermosetting resins thermosetting resins
  • cyclic olefin polymers cyclic olefin copolymers
  • glass and the like.
  • Materials constituting members other than the oxygen permeable layer (Ia), the gas barrier layer (II-a), and the laminated region of the culture surface of the culture vessel ( ⁇ ) are the materials constituting the oxygen permeable layer (Ia). is preferably the same as
  • At least part of the bottom member has at least an oxygen permeable layer (Ib) and a gas barrier layer (II-b), and the gas barrier layer (II-b) is oxygen-permeable. It is provided under the transmission layer (Ib).
  • the culture vessel ( ⁇ ) Since the culture vessel ( ⁇ ) has a gas barrier layer (II-b), oxygen permeation into the interior of the vessel is suppressed.
  • the oxygen permeable layer (Ib) When the oxygen permeable layer (Ib) is exposed by removing the gas barrier layer (II-b) from the oxygen permeable layer (Ib), the culture vessel ( ⁇ ) allows sufficient oxygen permeation into the vessel interior. becomes.
  • the bottom member has at least an oxygen permeable layer (Ib) and a gas barrier layer (II-b)
  • at least a portion of the bottom member has It means a region having both the oxygen permeable layer (Ib) and the gas barrier layer (II-b), and the entire region of the bottom member has the oxygen permeable layer (Ib) and the gas barrier layer (II-b).
  • II-b) or only a partial region of the bottom member may be a region having the oxygen permeable layer (Ib) and the gas barrier layer (II-b).
  • the region of the bottom member other than the region having both layers is The region is preferably composed of the oxygen permeable layer (b), and all regions other than the region having both layers in the bottom member are regions composed of the oxygen permeable layer (Ib). is more preferred.
  • the bottom usually includes the culture surface, so at least part of the culture surface is composed of the oxygen permeable layer (Ib) and the gas barrier layer (II-b). is a region having When at least part of the culture surface is a region having an oxygen permeable layer (Ib) and a gas barrier layer (II-b), it is easy to regulate oxygen supplied to the medium through the region.
  • the entire culture surface of the culture vessel ( ⁇ ) is preferably a region having an oxygen permeable layer (Ib) and a gas barrier layer (II-b).
  • the ratio of the area having the oxygen permeable layer (Ib) and the gas barrier layer (II-b) to the area of the culture surface is not particularly limited, but is preferably 80% or more, more preferably 90% or more, and still more preferably. is 95% or more.
  • the ratio of the area having the oxygen permeable layer (Ib) and the gas barrier layer (II-b) to the area of the culture surface is within the above range, the amount of oxygen supplied to cells and the like can be easily adjusted.
  • the gas barrier layer (II-b) is superimposed under the oxygen permeable layer (Ib). That is, the oxygen permeable layer (Ib) and the gas barrier layer (II-b) are stacked in the order of the oxygen permeable layer (Ib) and the gas barrier layer (II-b) from the inner side of the culture vessel ( ⁇ ). It is The gas barrier layer (II-b) may be directly overlaid under the oxygen permeable layer (Ib), or may be placed between the oxygen permeable layer (Ib) and the gas barrier layer (II-b). (eg, a metal deposition layer, the adhesive layer described above, etc.) may be included. The other layers may be stacked in the order of the oxygen permeable layer (Ib), the gas barrier layer (II-b), and other layers from the inside of the culture vessel ( ⁇ ).
  • the region of the oxygen permeable layer (Ib) and the region of the gas barrier layer (II-b) may have the same size, or one of them may be larger. Further, the area of the gas barrier layer (II-b) is preferably equal to or larger than the area of the oxygen permeable layer (Ib), since the gas barrier layer (II-b) can be easily attached and detached.
  • the area of the gas barrier layer (II-b) is preferably larger than the area of the culture surface because it is easy to adjust the oxygen supply amount and to attach and detach the gas barrier layer (II-b). ) is provided on the entire bottom surface of the culture vessel.
  • the area of the gas barrier layer (II-b) is preferably larger than the total bottom area of each well, and the gas barrier layer (II-b) is the entire bottom surface of the plate. It is preferable to provide
  • a material constituting members other than the bottom member (for example, side wall members) of the culture vessel ( ⁇ ) is not particularly limited, and known materials can be used. Such materials include, for example, polystyrene (PS), polydimethylsiloxane (PDMS), thermosetting resins, cyclic olefin polymers, cyclic olefin copolymers, glass, and the like.
  • PS polystyrene
  • PDMS polydimethylsiloxane
  • thermosetting resins thermosetting resins
  • cyclic olefin polymers cyclic olefin copolymers
  • glass glass
  • the material constituting the region of the bottom member of the culture vessel ( ⁇ ) other than the region having the oxygen permeable layer (Ib) and the gas barrier layer (II-b) is also not particularly limited, and known materials can be used. can.
  • Such materials include, for example, polystyrene (PS), polydimethylsiloxane (PDMS), thermosetting resins, cyclic olefin polymers, cyclic olefin copolymers, glass, and the like.
  • PS polystyrene
  • PDMS polydimethylsiloxane
  • thermosetting resins thermosetting resins
  • cyclic olefin polymers cyclic olefin copolymers
  • glass and the like.
  • the material constituting the region of the bottom member of the culture vessel ( ⁇ ) other than the region having the oxygen permeable layer (Ib) and the gas barrier layer (II-b) constitutes the oxygen permeable layer (Ib). It is preferably the same as the material.
  • the total light transmittance of the region having the oxygen permeable layer (Ib) and the gas barrier layer (II-b) of the bottom member of the culture vessel ( ⁇ ) is preferably 70.0% or more, more preferably 90.0%. 0% or more, more preferably 92.0% or more. Although there is no particular upper limit for the total light transmittance, it is usually 99.9%. It can be said that the fact that the total light transmittance is within the above range means that the bottom member has excellent transparency.
  • the total light transmittance of the regions of the oxygen permeable layer (Ib) and the gas barrier layer (II-b) of the bottom member of the culture vessel ( ⁇ ) is within the above upper and lower limits, the bottom member has excellent transparency. Therefore, it is easy to observe cells and the like with the naked eye and under a microscope.
  • the total light transmittance of the region having the oxygen permeable layer (Ib) and the gas barrier layer (II-b) of the bottom member of the culture vessel ( ⁇ ) is, for example, the oxygen permeable layer (Ib) and the gas barrier layer. It can be adjusted by adjusting the thickness of (II-b) or the materials constituting the oxygen permeable layer (Ib) and gas barrier layer (II-b).
  • the adhesive material constituting the adhesive layer is not particularly limited as long as it can form an adhesive layer having desired adhesiveness. Anything that is used can be used.
  • an adhesive can be used as the adhesive material.
  • the adhesive include acrylic adhesives, epoxy adhesives, silicone adhesives, and the like.
  • a conventionally known sealant resin can be used for the adhesive material.
  • the sealant resin is, for example, polyethylene such as low-density polyethylene (LDPE), linear low-density polyethylene (LLDPE), high-density polyethylene (HDPE), acid-modified polyethylene, polypropylene (PP), acid-modified polypropylene, and propylene.
  • Polymers ethylene-vinyl acetate copolymers, polypropylene-vinyl acetate copolymers, ethylene-(meth)acrylic acid ester copolymers, ethylene-(meth)acrylic acid copolymers, and metal crosslinked products thereof ionomers polyolefin resins, polyvinyl chloride resins, silicone resins, and the like, among which polyolefin resins are preferred from the viewpoint of low-temperature sealability, and polyethylene is particularly preferred because it is inexpensive.
  • the adhesive material may be used singly or in combination of two or more.
  • the thickness of the adhesive layer may be set as long as it has the desired adhesiveness, and can be appropriately set according to the constituent materials and the like.
  • the thickness of the adhesive layer can be in the range of 0.1 ⁇ m to 200 ⁇ m, preferably 5 ⁇ m to 100 ⁇ m, more preferably 10 ⁇ m to 60 ⁇ m, particularly preferably 15 ⁇ m to 40 ⁇ m. Within this range, the adhesive layer can be made excellent in workability and heat-sealability.
  • the method of manufacturing the culture vessel ( ⁇ ) or the culture vessel ( ⁇ ) is not particularly limited, nor is the equipment used for manufacturing.
  • a film or sheet containing the material that constitutes the oxygen-permeable layer (Ia) is used.
  • the gas barrier layer (II-a) is laminated to produce the culture vessel ( ⁇ ).
  • the culture vessel ( ⁇ ) is formed by directly molding a material that constitutes the oxygen permeable layer (Ia) by a method such as extrusion molding, solution casting molding, injection molding, blow molding, etc., and then forming the gas barrier layer (II- It can also be obtained by laminating a).
  • the material that constitutes the oxygen-permeable layer (I) is After obtaining a joint member by appropriately joining the film or sheet containing the can be obtained.
  • the joining method is not particularly limited, and the member containing the material constituting the oxygen-permeable layer (I) may be fusion-welded with another member, or they may be brought into close contact with each other via an adhesive or pressure-sensitive adhesive.
  • a film or sheet obtained by forming a film or sheet containing a material constituting the oxygen permeable layer (I) and a film or sheet containing a material constituting the gas barrier layer (II) and then laminating the two films or sheets. is formed, and the laminated film or sheet and other members are appropriately joined to obtain the culture vessel ( ⁇ ) or the culture vessel ( ⁇ ).
  • the culture vessel ( ⁇ ) when the bottom member and the side wall member contain the material constituting the oxygen permeable layer (Ib), for example, a film or sheet containing the material constituting the oxygen permeable layer (Ib) is used. After forming the film or sheet into a desired shape as necessary, the gas barrier layer (II-b) is adhered to produce the culture vessel ( ⁇ ).
  • the culture vessel ( ⁇ ) is directly molded into a desired shape by a method such as extrusion molding, solution casting molding, injection molding, blow molding, etc., using the material that constitutes the oxygen permeable layer (Ib). , can also be obtained by applying a gas barrier layer (II-b).
  • the method for manufacturing the culture vessel ( ⁇ ) may include a step of attaching the bottom member to the side wall member.
  • the culture vessel ( ⁇ ) is prepared by attaching a film or sheet containing a material that constitutes the oxygen permeable layer (Ib) to the side wall member to join the bottom member and the side wall member, and then the film or sheet. It can be produced by further attaching a film or sheet containing a material constituting the gas barrier layer (II-b) underneath.
  • a film or sheet containing the material constituting the gas barrier layer (II-b) is attached in advance under the film or sheet containing the material constituting the oxygen permeable layer (Ib). Then, by attaching the two attached films or sheets to the side wall member, the bottom member and the side wall member can be joined to manufacture.
  • the film or sheet may be produced by a solution casting method in which the material is dissolved in a solvent, poured onto a resin or metal, slowly dried while being leveled, and formed into a film (sheet).
  • the solvent used is not particularly limited, but hydrocarbon solvents such as cyclohexane, hexane, decane and toluene may be used. Two or more solvents may be mixed in consideration of the solubility and drying efficiency of the material.
  • a polymer solution is applied by a method such as table coating, spin coating, dip coating, die coating, spray coating, bar coating, roll coating, or curtain flow coating, dried, and peeled off to form a film or sheet.
  • the method of detachably stacking from (Ib) is not particularly limited, and known methods can be used. For example, fixing by magnetic force, fixing by adhesion, mechanical engagement, clamping by elastic spring members, tightening by screw members, and the like can be mentioned. Among these, fixing by adhesion is preferable because the gas barrier layer (II) is stably laminated or superimposed on the oxygen-permeable layer (I) and attachment and detachment are easy.
  • the gas barrier layer (II) 90% or more, more preferably 95% or more, and still more preferably 98% or more of the region laminated or superimposed on the oxygen-permeable layer (I) can be removed from the oxygen-permeable layer (I). preferable.
  • the ratio of the area of the removable gas barrier layer (II) to the area of the gas barrier layer (II) laminated or overlaid on the oxygen permeable layer (I) is within the above range, the amount of oxygen supplied to cells and the like is reduced. easier to adjust.
  • the removal of the gas barrier layer (II) from the oxygen permeable layer (I) may be performed at once or may be performed in multiple steps, and the gas barrier layer (II) may be removed from the oxygen permeable layer (I).
  • the area of the gas barrier layer (II) that can be removed relative to the area of the gas barrier layer (II) is a numerical value obtained by accumulating multiple times of removal.
  • the timing of removing the gas barrier layer (II) from the oxygen-permeable layer (I) is not particularly limited, as long as an appropriate timing can be determined depending on the type of cells and the culture conditions. This is the timing after it is confirmed with a microscope that the cells etc. have adhered to the culture vessel, or the timing at which the cells etc. are expected to adhere to the culture vessel.
  • the gas barrier layer (II-b) may be entirely removed from the oxygen permeable layer (Ib), or only part of the region of the gas barrier layer (II-b) may be removed from the oxygen permeable layer (Ib). ) can be removed. That is, part or all of the region of the gas barrier layer (II-b) may be removable from the oxygen permeable layer (Ib).
  • the gas barrier layer (II-b) is preliminarily provided with grid-like cuts, and only the gas barrier layer (II-b) corresponding to the wells to which sufficient oxygen is to be supplied to the cells is removed.
  • the peel strength of the gas barrier layer (II-b) to the oxygen permeable layer (Ib) is preferably 0.01 to 0.20 N/25 mm, more preferably 0.02 to 0.15 N/25 mm. .
  • the peel strength is at least the above lower limit, it is possible to prevent unintended peeling, for example, peeling of the gas barrier layer (II-b) from the oxygen permeable layer (Ib) by its own weight.
  • the peel strength is equal to or less than the above upper limit, it is possible to prevent damage to cells and the like due to impact during peeling, for example, damage to cells and the like caused by vibration due to the sound of peeling or vibration of the hand holding the culture vessel. .
  • the peel strength can be measured by the following method.
  • a test piece with a width of 25 mm prepared from the gas barrier layer (II-b) is attached to the upper surface of the oxygen-permeable layer (Ib) with the surface to be measured for peel strength facing downward, and a 2 kg roller is reciprocated once. After holding this for 30 minutes in an environment of 23 ° C. and RH 50%, using a peel tester, in accordance with JIS Z 0237, in an environment of 23 ° C. and RH 50%, a peel angle of 180 ° and a tensile speed of 10 mm / min.
  • the peel strength [N/25 mm] is measured under the conditions of As the peel tester, for example, Strograph ES manufactured by Toyo Seiki Seisakusho Co., Ltd. can be used. Perform the test three times and calculate the average value.
  • a method for culturing a cell, tissue, or organ which is one aspect of the present invention, comprises a step (Aa) of culturing a cell, tissue, or organ using a culture vessel ( ⁇ ); A step (Ba) of removing the gas barrier layer (II-a) from the oxygen permeable layer (Ia), and using the culture vessel obtained in the step (Ba), cells, tissues, or A step (Ca) of further culturing the organ is included.
  • a method for culturing a cell, tissue, or organ comprises a step (Ab) of culturing a cell, tissue, or organ using a culture vessel ( ⁇ ); The step (Bb) of removing the gas barrier layer (II-b) from the oxygen permeable layer (Ib), and using the culture vessel obtained in the step (Bb), cells, tissues, or A step (Cb) of further culturing the organ is included.
  • Step (Aa) or step (Ab) is a step of culturing cells, tissues, or organs using a culture vessel ( ⁇ ) or a culture vessel ( ⁇ ).
  • the culture vessel ( ⁇ ) or the culture vessel ( ⁇ ) preferably has a water contact angle of the oxygen permeable layer (I) of 30 to 120°.
  • the oxygen-permeable layer (I) preferably contains a 4-methyl-1-pentene polymer (X).
  • the 4-methyl-1-pentene polymer (X) is a 4-methyl-1-pentene homopolymer (x1) and 4-methyl-1-pentene
  • At least one polymer selected from a copolymer (x2) of at least one olefin selected from ethylene and ⁇ -olefins having 3 to 20 carbon atoms (excluding 4-methyl-1-pentene) is preferably
  • the culture vessel ( ⁇ ) or the culture vessel ( ⁇ ) is coated with a natural polymeric material, a synthetic polymeric material, or an inorganic material on the culture surface, preferably the side of the culture surface that comes into contact with the medium and/or the cells. preferably.
  • the gas barrier layer (II) preferably contains polyethylene terephthalate (PET).
  • a method for culturing cells or the like in a medium is not particularly limited, and may be carried out according to a known protocol, and commercially available medium and culture kits may be used.
  • the medium used for culture is not particularly limited as long as it allows cells to proliferate, and may be appropriately selected according to the cell type used.
  • the amount of medium used for culture and the frequency of medium exchange are not particularly limited.
  • the culture temperature is not particularly limited, it is usually carried out at about 25-40°C.
  • the culture period in the step (Aa) or step (Ab) is not particularly limited, and the culture may be continued until the cells and the like adhere sufficiently to the culture vessel.
  • the culture period is preferably 12 to 48 hours, more preferably 18 to 36 hours, still more preferably 20 to 30 hours.
  • the initial culture is performed in a state of low oxygen supply, so the adhesiveness tends to be better.
  • Step (Ba) or step (Bb) is a step of removing the gas barrier layer (II) from the oxygen permeable layer (I). Since the culture vessel ( ⁇ ) has the gas barrier layer (II), permeation of oxygen into the interior of the vessel is suppressed. is exposed, the culture vessel ( ⁇ ) or the culture vessel ( ⁇ ) is sufficiently oxygen-permeable to the interior of the vessel.
  • the method for removing the gas barrier layer (II) from the oxygen permeable layer (I) is not particularly limited, and a suitable method may be used according to the method for fixing the gas barrier layer (II) to the oxygen permeable layer (I).
  • Step (Ba) or step (Bb) is not particularly limited, but it is preferable to perform it after the cells etc. have sufficiently adhered to the culture vessel.
  • Step (B-a) or step (B-b) can be carried out preferably 12 to 48 hours, more preferably 18 to 36 hours, still more preferably 20 to 30 hours after seeding cells or the like. preferable.
  • Step (Ca) or step (Cb) is a step of further culturing cells, tissues, or organs using the culture vessel obtained in step (Ba) or step (Bb). is.
  • the method of culturing cells or the like in a medium is not particularly limited, and can be carried out according to step (Aa) or step (Ab).
  • the medium used for culture, the amount of medium used for culture, and the frequency of medium exchange may be the same as or different from those in step (Aa) or step (Ab).
  • the culture period in step (Ca) or step (Cb) is not particularly limited, and the cells may be cultured until the desired number of cells is reached.
  • the culture period is preferably 12 to 48 hours, more preferably 18 to 36 hours, still more preferably 20 to 30 hours.
  • the cells and the like are cultured in a state in which oxygen is sufficiently supplied, so that the functions of the cells and the like are likely to be maintained normally.
  • TPX film 4-methyl-1-pentene polymer film (thickness 50 ⁇ m, manufactured by Mitsui Chemicals Tohcello Co., Ltd., trade name: Opulan X-88B)
  • PET film 1 A commercially available PET film made of a mixture of PET and an adhesive (thickness 80 ⁇ m, manufactured by BM Equipment Co., Ltd., trade name: qPCR plate seal (ABI, crimp type)
  • PET film 2 A commercially available PET film (Nichiei Shinka Co., Ltd., trade name: PET75-H270 (10)) consisting of a base layer (PET layer, thickness 75 ⁇ m) and an adhesive layer (thickness 10 ⁇ m).
  • PP film Commercially available PP film (Nichiei Shinka Co., Ltd., trade name: PP60-H270 (10)) consisting of a substrate layer (PET layer, thickness 60 ⁇ m) and an adhesive layer (thickness 10 ⁇ m)
  • PDMS vessel A commercially available 96-well culture vessel (product name: G-plate, manufactured by VECELL, model number: V96WGPB-10) whose bottom surface is composed of a PDMS (dimethylpolysiloxane) film with a thickness of 350 ⁇ m.
  • PS container a commercially available 24-well PS culture container made of PS (polystyrene) with a bottom thickness of 1000 ⁇ m (manufactured by Corning)
  • the oxygen permeability coefficient was measured using a differential pressure type gas permeability measuring device MT-C3 manufactured by Toyo Seiki Seisakusho Co., Ltd. under an environment of a temperature of 23° C. and a humidity of 0%.
  • the diameter of the measuring portion was 70 mm (transmission area was 38.46 cm 2 ). Since the oxygen permeability coefficient was expected to be large, the sample was masked with aluminum in advance to set the actual permeation area to 5.0 cm 2 .
  • the total light transmittance of the measurement sample was measured using a haze meter (NDH2000, manufactured by Nippon Denshoku Industries Co., Ltd.) in accordance with JIS K 7361-1.
  • the water contact angle was measured according to Japanese Industrial Standards JIS-R3257 (testing method for wettability of substrate glass surface). Under constant temperature and humidity conditions of 25 ⁇ 5 ° C and 50 ⁇ 10%, a water droplet of a volume of 4 ⁇ L or less that can be regarded as a spherical water droplet is dropped on the surface of the measurement sample. The angle of the contact interface between the measurement sample and the water droplet was measured within 1 minute from immediately after the contact.
  • a 0.1 M hydrochloric acid solution (for volumetric analysis, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) is diluted 100 times with water for injection (Japanese Pharmacopoeia, manufactured by Otsuka Pharmaceutical Co., Ltd.) to prepare a 0.001 M hydrochloric acid solution. Sterilized by filtration. A 3 mg/mL collagen solution (Cell Matrix Type IP, derived from porcine tendon, manufactured by Nitta Gelatin Co., Ltd.) was diluted 6-fold with a 0.001 M hydrochloric acid solution to prepare a 0.5 mg/mL collagen solution.
  • a medium (A) was added to a centrifuge tube (50 mL) containing a cell suspension containing rat primary frozen hepatocytes.
  • Medium (A) is 1.5 mL of fetal bovine serum (FBS, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) diluted to 3.0 g/mL with water for injection (manufactured by Fuso Pharmaceutical Co., Ltd.).
  • FBS fetal bovine serum
  • epidermal growth factor EGF, for cell biology, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.
  • insulin solution 10 mg / mL in HEPESS, manufactured by Sigma-Aldrich Japan LLC
  • penicillin-streptomycin solution (containing 5000 units/mL penicillin, 5000 ⁇ g/mL streptomycin, for culture, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) is 0.3 mL
  • D -MEM medium (4500 mg/mL D-glucose, 584 mg/mL L-glutamine, 15 mg/mL phenol red, 110 mg/mL sodium pyruvate, 3700 mg/mL sodium bicarbonate containing, for culture, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd. ) was added to 13 mL.
  • the cell density was adjusted by adjusting the number of cells in a cell suspension
  • the amount of protein was determined by adding 200 ⁇ L of PBS( ⁇ ) to the medium, collecting the cells in an Eppendorf tube using a cell scraper, and centrifuging (4° C., 22000 ⁇ g, 10 minutes). Thereafter, the supernatant was removed, 100 ⁇ L of 0.1 M sodium hydroxide solution was added, and the protein amount was measured using Pierce TM BCA Protein Assay Kit (manufactured by Thermo Fisher Scientific). Absorbance at a wavelength of 450 nm was measured with a plate reader (SPECTRA max PLUS384, manufactured by Molecular Devices).
  • the amount of metabolic activity (pmol/L) of the Luciferin-CEE solution obtained with a luminometer was measured using P450-Glo TM CYP1A1 Assay kit (manufactured by Promega), and the protein amount and Luciferin-CEE obtained from the absorbance were measured.
  • the metabolic activity value (pmol/min/mg protein) was calculated by dividing by the reaction time of the solution. A representative 3 wells were measured and the average value was calculated.
  • a medium (B) was added to a centrifuge tube (50 mL) containing a cell suspension containing human primary frozen hepatocytes.
  • Medium (B) contains 2.5 mL of fetal bovine serum (Fetal Bovine Serum, FBS, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), Antibiotic-Antimycotic (10000 units/mL penicillin, 10000 ⁇ g/mL streptomycin, 25 ⁇ g/mL amphotericin B) , Gibco TM ) diluted to 1 ⁇ 10 ⁇ 3 M with ethanol (for molecular biology, manufactured by FUJIFILM Wako Pure Chemical Industries, Ltd.) and dexamethasone (for biochemistry, manufactured by FUJIFILM Wako Pure Chemical Industries, Ltd.).
  • 0.05 mL of solution 0.02 mL of insulin solution (10 mg/mL in HEPESS, manufactured by Sigma-Aldrich Japan LLC), 0.5 mL of GlutaMAX solution (Gibco TM ), HEPES buffer solution (biochemical buffer, Dojindo Co., Ltd.), 0.05 mL of trisodium L-ascorbic acid 2-phosphate (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) adjusted to 100 mM with water for injection, and 1.0 mg/mL with water for injection.
  • EGF epidermal growth factor
  • BSA Bactet al.
  • William's E Medium 2000 mg/mL D-glucose, 10 mg/mL phenol red, 25 mg/mL sodium pyruvate, 2200 mg/mL sodium bicarbonate, for culture, Giboco
  • the cell density was adjusted in the same manner as the method for adjusting the cell number of the cell suspension containing rat primary frozen hepatocytes, and cultured at a cell density of 1.0 ⁇ 10 5 cells/cm 2 .
  • Medium (C) contains 2.5 mL of fetal bovine serum (Fetal Bovine Serum, FBS, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), Antibiotic-Antimycotic (10000 units/mL penicillin, 10000 ⁇ g/mL streptomycin, 25 ⁇ g/mL amphotericin B) , Gibco TM ) diluted to 1 ⁇ 10 ⁇ 3 M with ethanol (for molecular biology, manufactured by FUJIFILM Wako Pure Chemical Industries, Ltd.) and dexamethasone (for biochemistry, manufactured by FUJIFILM Wako Pure Chemical Industries, Ltd.).
  • FBS Fetal Bovine Serum
  • Antibiotic-Antimycotic 10000 units/mL penicillin, 10000 ⁇ g/mL streptomycin, 25 ⁇ g/mL amphotericin B
  • Gibco TM Gibco TM diluted to 1 ⁇ 10 ⁇ 3 M with ethanol (for molecular biology, manufactured by FUJIFILM Wa
  • EGF epidermal growth factor
  • EGF epidermal growth factor
  • Gibco TM HepExtended Supplement
  • the film forming the bottom surface of the culture vessel and the peeled film (either PET film 1, 2 or PP film) were checked for breakage, etc., and evaluated according to the following criteria.
  • AA The film forming the bottom surface of the culture vessel and the peeled film (either PET film 1, 2 or PP film) were not torn, and no warping of the film forming the bottom surface of the culture vessel was observed, and the peelability was good. there were.
  • BB The film forming the bottom surface of the culture vessel and/or the peeled film (either PET film 1, 2 or PP film) was torn, or the film forming the bottom surface of the culture vessel was bent and the peelability was poor. rice field.
  • Example 1 The TPX film was plasma-treated using an atmospheric pressure plasma surface treatment apparatus (manufactured by Sekisui Chemical Co., Ltd.), filling the chamber with a stream of nitrogen (treatment speed: 2 m/min, output: 4.5 kW, 2 reciprocations).
  • the plasma-treated TPX film was cut into a size of 8 cm ⁇ 12 cm, and adhered to a bottomless PS 24-well container frame via a medical adhesive (manufactured by 3M), and the bottom of the culture plate was composed of the TPX film.
  • a 24-well culture plate was prepared.
  • a rubber roller (roller width: 45 mm, roller mass: 2 kg) is reciprocated twice to press the PET film 1 against the TPX film on the bottom of the culture plate on the side opposite to the container frame, and the culture plate is pressed at a constant pressure.
  • a 24-well culture plate whose bottom surface was composed of a laminate of TPX film and PET film 1 was prepared. That is, the surface of the PET film 1 from which the release paper was removed was adhered to the lower surface of the TPX film constituting the bottom surface of the culture plate. Thereafter, the 24-well culture plate was packed in a gamma-ray resistant bag and irradiated with 10 kGy of gamma rays for sterilization.
  • a medium (0.5 mL) containing rat primary frozen hepatocytes was added to A to D of the A to E culture vessels (1) prepared above with a micropipette, and the concentration was 1.0 ⁇ 10 5 cells/cm.
  • the cells were seeded at a cell density of 2 and cultured at 37° C. under 5% CO 2 . Twenty-four hours after seeding the cells, the culture vessels AD were removed from the incubator.
  • the culture vessels (1) A and B were returned to the incubator after peeling off the bottom PET film, and cultured for another 24 hours.
  • the culture vessel (1) C the dissolved oxygen concentration in the medium was measured.
  • the metabolic activity value was measured as an index of the normal function of hepatocytes.
  • the culture vessel (1) A from which the PET film 1 was removed was removed from the incubator, and the dissolved oxygen concentration was measured.
  • the culture vessel (1)B from which the PET film 1 was removed was removed from the incubator, and the metabolic activity value was measured. Table 1 shows the results.
  • a medium (0.5 mL) containing human primary frozen hepatocytes was added to the culture vessel (1) E prepared above with a micropipette and seeded at a cell density of 1.0 ⁇ 10 5 cells/cm 2 . Cultivation was started at 5% CO 2 at 0°C. Twenty-four hours after seeding, the culture vessel (1) E was taken out from the incubator, the PET film 1 on the bottom surface was peeled off, and peelability evaluation and cell adhesion evaluation were performed. The results are shown in Table 1 and FIG.
  • Example 2 The PET film 1 on the bottom of the culture vessel (1) was changed to a PP film, and the surface treatment of the TPX film was corona treated using a table-type corona treatment device (manufactured by Kasuga Denki) (treatment speed 3 m / min,
  • a table-type corona treatment device manufactured by Kasuga Denki
  • rat frozen hepatocytes and human frozen hepatocytes were cultured and evaluated in the same manner as in Example 1, except that the output was changed to 0.5 kW, two reciprocations). Table 1 shows the results.
  • Example 3 Rat frozen hepatocytes and human frozen hepatocytes were cultured and evaluated in the same manner as in Example 1 except that the PET film 1 on the bottom of the culture vessel (1) was changed to PET film 2.
  • Example 3 and Table 1 shows the results.
  • Example 4 A pressure-bonding rubber roller (roller width: 45 mm, roller mass: 2 kg) was reciprocated twice to press the PET film 2 against the PDMS film constituting the bottom surface of the PDMS container at a constant pressure, so that the bottom surface of the culture plate was the PDMS film.
  • a 96-well culture plate composed of a laminate of PET film 2 and PET film 2 was prepared, 0.1 mL of 0.5 mg/mL collagen solution was added to each well, and excess collagen solution was removed. After standing at room temperature for 30-60 minutes, it was washed with Dulbecco's PBS(-) and dried overnight at room temperature.
  • Example 4 rat frozen hepatocytes and human frozen hepatocytes were cultured and evaluated in the same manner as in Example 1. Table 1 shows the results.
  • Reference Example 1 was prepared by culturing and evaluating rat frozen hepatocytes in the same manner as in Example 1, except that the PET film 1 on the bottom surface of the culture vessel (1) was not peeled off. Table 2 shows the results. Human frozen hepatocytes were not cultured or evaluated.
  • Comparative Example 1 In Comparative Example 1, a commercially available 24-well PS culture vessel (PS) was used instead of the 24-well culture plate whose bottom surface was composed of a laminate of TPX film and PET film 1. Culture and evaluation of rat frozen hepatocytes were performed in the same manner as in 1. Table 2 shows the results. Human frozen hepatocytes were not cultured or evaluated.
  • PS PS culture vessel
  • Comparative Example 2 rat frozen hepatocytes and human frozen liver were obtained in the same manner as in Example 1 except that the bottom surface of the culture plate used a 24-well culture plate composed of a TPX film and the PET film 1 was not attached. Cell culture and evaluation were performed. The results are shown in Table 2 and FIG.
  • Comparative Example 1 had a low metabolic activity maintenance rate, and the metabolic activity of cells decreased from 24 hours after seeding to 48 hours after seeding. This is probably because the bottom surface of the culture vessel was made of PS with low oxygen permeability, and oxygen supply to the cells was insufficient from 24 hours after seeding to 48 hours after seeding.
  • Comparative Example 2 had a good rate of maintaining metabolic activity, and was able to suppress the decrease in metabolic activity of cells that occurred from 24 hours after seeding to 48 hours after seeding. However, 24 hours after seeding, the cells did not adhere to the culture vessel and were detached. This is probably because the bottom surface of the culture vessel was composed only of a TPX film with high oxygen permeability, and the oxygen supply to the cells was excessive from immediately after seeding to 24 hours after seeding.

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