WO2023085019A1 - 培養容器及び培養方法 - Google Patents
培養容器及び培養方法 Download PDFInfo
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- 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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- culture vessel
- oxygen
- gas barrier
- barrier layer
- culture
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Images
Classifications
-
- 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
- C12M3/00—Tissue, human, animal or plant cell, or virus culture apparatus
-
- 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
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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Abstract
Description
本発明の態様は、例えば以下の〔1〕~〔7〕に関する。
〔1〕 細胞、組織、又は器官を培地中で培養する培養容器であって、前記培養容器の培養面の少なくとも一部が、下記要件(1)及び(2)を満たす酸素透過層(I-a)と、下記要件(3)及び(4)を満たすガスバリア層(II-a)との積層領域を有し、前記ガスバリア層(II-a)が前記酸素透過層(I-a)の下面に設けられ、前記ガスバリア層(II-a)が前記酸素透過層(I-a)から培養中に着脱可能である、培養容器。
(1)温度23℃、湿度0%の時の酸素透過度P(I-a)が20000~60000cm3/(m2×24h×atm)である
(2)JIS K 7361-1に準拠して測定した全光線透過率が70%以上である
(3)温度23℃、湿度0%の時の酸素透過度P(II-a)が3000cm3/(m2×24h×atm)以下である
(4)JIS K 7361-1に準拠して測定した全光線透過率が70%以上である
〔2〕 前記酸素透過層(I-a)の水接触角が、30~120°である、〔1〕に記載の培養容器。
〔3〕 前記酸素透過層(I-a)が、4-メチル-1-ペンテン重合体(X)を含む、〔1〕又は〔2〕に記載の培養容器。
〔4〕 前記4-メチル-1-ペンテン重合体(X)が、4-メチル-1-ペンテン単独重合体(x1)並びに、4-メチル-1-ペンテンと、エチレン及び炭素数3~20のα-オレフィン(4-メチル-1-ペンテンを除く)から選ばれる少なくとも1種のオレフィンとの共重合体(x2)から選択される少なくとも1種の重合体である、〔3〕に記載の培養容器。
〔5〕 培養面に、天然高分子材料、合成高分子材料、又は無機材料がコーティングされた、〔1〕~〔4〕のいずれかに記載の培養容器。
〔6〕 前記ガスバリア層(II-a)が、ポリエチレンテレフタレート(PET)を含む、〔1〕~〔5〕のいずれかに記載の培養容器。
〔7〕 細胞、組織、又は器官の培養方法であって、〔1〕~〔6〕のいずれかに記載の培養容器を用いて、細胞、組織、又は器官を培養する工程(A-a)、前記ガスバリア層(II-a)を前記酸素透過層(I-a)から取り外す工程(B-a)、及び前記工程(B-a)で得られた培養容器を用いて、細胞、組織、又は器官をさらに培養する工程(C-a)、を含む、培養方法。
〔i〕 細胞、組織、又は器官を培養する培養容器であって、前記培養容器が、底面部材と、側壁部材とを有し、前記底面部材と前記側壁部材とが接合することで少なくとも一つの培養空間が形成され、前記底面部材の少なくとも一部が、少なくとも酸素透過層(I-b)と、ガスバリア層(II-b)とを有し、前記ガスバリア層(II-b)が、前記酸素透過層(I-b)の下に、前記酸素透過層(I-b)から着脱可能に重ねられ、前記ガスバリア層(II-b)が、下記要件(5)を満たし、前記酸素透過層(I-b)が、下記要件(6)を満たす、培養容器。
(5)温度23℃、湿度0%の時の酸素透過度P(II-b)が3000cm3/(m2×24h×atm)以下である
(6)温度23℃、湿度0%の時の酸素透過度P(I-b)が前記P(II-b)の6.0倍以上である
〔ii〕 前記ガスバリア層(II-b)が、ポリエチレンテレフタレート(PET)およびポリオレフィンからなる群から選択される少なくとも1種を含み、かつ、下記要件(7)または(8)を満たす、〔i〕に記載の培養容器。
(7)ポリエチレンテレフタレート(PET)およびポリオレフィンからなる群から選択される少なくとも1種と、粘着剤との混合物を含む
(8)ポリエチレンテレフタレート(PET)およびポリオレフィンからなる群から選択される少なくとも1種を含む層と、粘着剤を含む層と、を有する
〔iii〕 下記方法で測定される、前記ガスバリア層(II-b)の前記酸素透過層(I-b)に対する剥離強度が、0.01~0.20N/25mmである、〔i〕又は〔ii〕に記載の培養容器。
[幅25mmのガスバリア層(II-b)を酸素透過層(I-b)に貼付した後、剥離試験機を用いて、ガスバリア層(II-b)を酸素透過層(I-b)から剥離角度180°で10mm/分の速度で剥離する試験を行う。]
〔iv〕 前記酸素透過層(I-b)が、4-メチル-1-ペンテン重合体(X)を含む、〔i〕~〔iii〕のいずれかに記載の培養容器。
〔v〕 前記4-メチル-1-ペンテン重合体(X)が、4-メチル-1-ペンテン単独重合体(x1)並びに、4-メチル-1-ペンテンと、エチレン及び炭素数3~20のα-オレフィン(4-メチル-1-ペンテンを除く)から選ばれる少なくとも1種のオレフィンとの共重合体(x2)から選択される少なくとも1種の重合体である、〔iv〕に記載の培養容器。
〔vi〕 前記P(I-b)が、20000~60000cm3/(m2×24h×atm)である、〔i〕~〔v〕のいずれかに記載の培養容器。
〔vii〕 前記底面部材の、前記酸素透過層(I-b)と前記ガスバリア層(II-b)とを有する領域の、JIS K 7361-1に準拠して測定した全光線透過率が70%以上である、〔i〕~〔vi〕のいずれかに記載の培養容器。
〔viii〕 前記酸素透過層(I-b)の、JIS K 7361-1に準拠して測定した全光線透過率が70%以上である、〔i〕~〔vii〕のいずれかに記載の培養容器。
〔ix〕 前記ガスバリア層(II-b)の、JIS K 7361-1に準拠して測定した全光線透過率が70%以上である、〔i〕~〔viii〕のいずれかに記載の培養容器。
〔x〕 前記酸素透過層(I-b)の水接触角が、30~120°である、〔i〕~〔ix〕のいずれかに記載の培養容器。
〔xi〕 前記底面部材の培養面に、天然高分子材料、合成高分子材料、又は無機材料がコーティングされた、〔i〕~〔x〕のいずれかに記載の培養容器。
〔xii〕 細胞、組織、又は器官の培養方法であって、〔i〕~〔xi〕のいずれかに記載の培養容器を用いて、細胞、組織、又は器官を培養する工程(A-b)、前記ガスバリア層(II-b)を前記酸素透過層(I-b)から取り外す工程(B-b)、及び前記工程(B-b)で得られた培養容器を用いて、細胞、組織、又は器官をさらに培養する工程(C-b)、を含む、培養方法。
〔xiii〕 〔i〕~〔xii〕のいずれかに記載の培養容器の製造方法であって、前記方法が、前記側壁部材に、前記底面部材を貼付する工程を含む、培養容器の製造方法。
なお、数値範囲に関する「A~B」との記載は、特に断りがなければ、A以上B以下であることを表す。例えば、「1~5%」との記載は、1%以上5%以下を意味する。
下記要件(1)及び(2)を満たす酸素透過層(I-a)と、
下記要件(3)及び(4)を満たすガスバリア層(II-a)との積層領域を有し、
前記ガスバリア層(II-a)が前記酸素透過層(I-a)の下面に設けられ、
前記ガスバリア層(II-a)が前記酸素透過層(I-a)から培養中に着脱可能である、培養容器である。この培養容器を培養容器(α)と称する。
(1)温度23℃、湿度0%の時の酸素透過度P(I-a)が20000~60000cm3/(m2×24h×atm)である
(2)JIS K 7361-1に準拠して測定した全光線透過率が70%以上である
(3)温度23℃、湿度0%の時の酸素透過度P(II-a)が3000cm3/(m2×24h×atm)以下である
(4)JIS K 7361-1に準拠して測定した全光線透過率が70%以上である
細胞、組織、又は器官を培養する培養容器であって、前記培養容器が、底面部材と、側壁部材とを有し、
前記底面部材と前記側壁部材とが接合することで少なくとも一つの培養空間が形成され、
前記底面部材の少なくとも一部が、少なくとも酸素透過層(I-b)と、ガスバリア層(II-b)とを有し、
前記ガスバリア層(II-b)が、前記酸素透過層(I-b)の下に、前記酸素透過層(I-b)から着脱可能に重ねられ、
前記ガスバリア層(II-b)が、下記要件(5)を満たし、
前記酸素透過層(I-b)が、下記要件(6)を満たす、培養容器である。この培養容器を、培養容器(β)と称する。
(5)温度23℃、湿度0%の時の酸素透過度P(II-b)が3000cm3/(m2×24h×atm)以下である
(6)温度23℃、湿度0%の時の酸素透過度P(I-b)が前記P(II-b)の6.0倍以上である。
本明細書において、細胞、組織、又は器官は、単に「細胞等」とも称する。細胞等の由来は、特に限定されず、動物、植物、昆虫、菌、原生生物、細菌などあらゆる生物であってよいが、動物、又は植物が好ましく、動物がさらに好ましく、特に哺乳動物が好ましい。本発明の一態様である培養容器は、細胞等の接着性に優れるので、細胞等は接着性のものであることが好ましい。培養容器の培養面での培養に適することから、細胞等は細胞であることが好ましい。本発明の一態様において、細胞等は、好気性であると好ましく、嫌気性のものを含まないことがより好ましい。
細胞は、浮遊性細胞であってもよく、接着性細胞であってもよいが、好ましくは接着性細胞である。
細胞は、2次元で培養される細胞であってもよいし、3次元で培養される細胞であってもよく、細胞を培養して得られるスフェロイドを含む。細胞は、初代培養細胞あるいは株化継代された細胞のいずれであってもよいが、好ましくは初代培養細胞である。細胞は、凍結又は再凍結したものを用いてもよい。
前記細胞は1種単独で用いてもよく、2種以上を組み合わせて用いてもよい。
また、細胞は、幹細胞または前駆細胞を含んでもよく、除いてもよい。細胞は、上皮系幹細胞または前駆細胞を除いてもよく、含んでいてもよい。
本発明の一態様において、培養とは、細胞等を増殖、維持させることだけでなく、細胞等の播種、継代、分化誘導、自己組織化誘導等のプロセスも含む広い意味で用いる。培養に用いる培地等は制限されず、細胞等の特性に応じた培地を選択すればよい。
細胞播種密度が上記の範囲であると、上記範囲外の場合と比べて、細胞が容器に良好に接着し、細胞の正常な機能が維持されやすい。
培養容器(α)において、培養容器とは、細胞、組織、又は器官を培地中で培養するために用いられる容器全てを意味する。培地中で培養するとは、細胞等の少なくとも一部が、培地に浸漬されて培養されるという意味であり、細胞等の全体が培地に浸漬されていなくてもよい。
培養容器(β)において、培養容器とは、細胞、組織、又は器官を培養するために用いられる容器全てを意味する。該培養は、培地中で行ってもよい。
培養容器は、少なくとも1つのウェルを有する培養容器であることが好ましく、少なくとも1つのウェルを有するプレートであることがさらに好ましく、6ウェル、12ウェル、24ウェル、48ウェル、96ウェル、384ウェル、1536ウェル等の複数のウェルを有するプレートであることがさらに好ましい。一般にウェルのようなくぼみ形状を底面に有する培養容器は、底面の複雑な形状を安定させるために底面を厚くする必要があり、細胞等への酸素供給が充分に行われ難い。培養容器(α)のように、底面が酸素透過層(I-a)とガスバリア層(II-a)との積層領域を有すると、1ウェル、6ウェル、12ウェル、24ウェル、48ウェル、96ウェル、384ウェル、1536ウェル等の複数のウェルを有するプレートであっても、形状が安定しており、細胞等への酸素供給量も調節しやすい。培養容器(β)のように、底面部材の少なくとも一部が、少なくとも酸素透過層(I-b)と、ガスバリア層(II-b)とを有し、前記ガスバリア層(II-b)が前記酸素透過層(I-b)の下に重ねられている場合も、同様に、複数のウェルを有するプレートであっても、形状が安定しており、細胞等への酸素供給量も調節しやすい。
底面部材と、側壁部材とは、培養空間内の細胞等または培地が漏れ出さないように接合している。接合は、機械的接合、材料的接合、接着接合のいずれであってもよいが、所望の材料からなる、底面部材と側壁部材の組み合わせが容易であることから、接着接合が好ましい。
ここで培養面とは、培養容器を構成する部分のうち、細胞等を培養する際に、培地及び/又は細胞等が接触している部分、若しくは培地及び/又は細胞等が接触する予定の部分を意味する。例えば、培養容器がディッシュ、フラスコ又はプレートの場合、通常、底面部分が培養面を含み、該培養面の上側、すなわち、培養容器の内側に培地及び/又は細胞等が接触する。
コーティングされた培養容器は、細胞等の接着性、増殖性がより優れる。これは、培養面にコーティングされている天然高分子材料、合成高分子材料、又は無機材料が、細胞等の足場となるためと考えられる。したがって、細胞等を接着させて培養する際には、培養容器は、培養面に、天然高分子材料、合成高分子材料、又は無機材料がコーティングされていることが好ましく、培養面の培地及び/又は細胞等が接触する側が、天然高分子材料、合成高分子材料、又は無機材料によりコーティングされていることがより好ましい。
表面改質処理に用いる方法は特に限定されないが、例えばコロナ処理、プラズマ処理、オゾン処理、紫外線処理等の親水化処理、エステル化、シリル化、フッ化等の疎水化処理、表面グラフト重合、化学蒸着、エッチング、又は、ヒドロキシル基、アミノ基、スルホン基、チオール基、カルボキシル基等の特定の官能基付加、シランカップリング、チタンカップリング、ジルコニウムカップリング等の特定の官能基による処理、酸化剤等による表面粗化、ラビングやサンドブラスト等の物理的処理等が挙げられる。これらの表面改質処理は、単独で行ってもよいし、2種以上を組み合わせて行ってもよい。
プラズマ処理を行う場合には、同伴させるガスとして、窒素、水素、ヘリウム、酸素、アルゴンなどが用いられ、好ましくは、窒素、ヘリウム、アルゴンから選択される少なくとも一種のガスが選ばれる。
以下で説明する、酸素透過層(I-a)及び酸素透過層(I-b)を総称して、酸素透過層(I)ともいう。
酸素透過層(I-a)は、下記要件(1)及び(2)を満たす層である。
(1)温度23℃、湿度0%の時の酸素透過度P(I-a)が20000~60000cm3/(m2×24h×atm)である
(2)JIS K 7361-1に準拠して測定した全光線透過率が70%以上である
酸素透過度P(I-a)は、具体的には以下の方法で測定できる。
酸素透過層(I-a)から測定サンプルを作製し、差圧式ガス透過率測定法により、温度23℃、湿度0%での酸素透過係数[cm3×mm/(m2×24h×atm)]を測定する。測定に用いる機器は差圧式ガス透過率測定法を用いたものであれば特に制限されないが、例えば株式会社東洋精機製作所製の差圧式ガス透過率測定装置MT-C3が挙げられる。測定サンプルは、酸素透過層(I-a)から厚さ50μmの90×90mmの試験片を切り出して作製し、測定部径は70mm(透過面積は38.46cm2)とすると好ましい。酸素透過度が大きいため、予めサンプルにアルミニウムマスクを施し、実透過面積を5.0cm2とすることがより好ましい。測定サンプルは、微細加工、表面改質処理を行ったものでもよいし、行っていないものでもよいが、何も処理を行っていないものが好ましい。酸素透過係数をフィルムの厚さ(μm)で除した値を酸素透過度[cm3/(m2×24h×atm)]とする。
酸素透過層(I-a)の酸素透過度P(I-a)は、例えば、酸素透過層(I-a)の厚さ、又は酸素透過層(I-a)を構成する材料等を調整することにより、調整することができる。
全光線透過率は、具体的には以下の方法で測定できる。
酸素透過層(I-a)から作製した試験片を使用して、JIS K 7361-1に準拠して、(株)村上色彩技術研究所製のヘイズ透過率計HM-150(D65光源)を用い、全透過光量を測定し、下記式にて全光線透過率を求める。
全光線透過率(%)=100×(全透過光量)/(入射光量)
酸素透過層(I-a)の全光線透過率は、例えば、酸素透過層(I-a)の厚さ、又は酸素透過層(I-a)を構成する材料等を調整することにより、調整することができる。
酸素透過層(I-b)は、下記要件(6)を満たす層である。
温度23℃、湿度0%の時の酸素透過度P(I-b)が、後述するP(II-b)の6.0倍以上である
酸素透過度P(I-b)は、酸素透過度P(I-a)と同様の方法及び好適態様で測定できる。
酸素透過層(I-b)の酸素透過度P(I-b)は、例えば、酸素透過層(I-b)の厚さ、又は酸素透過層(I-b)を構成する材料等を調整することにより、調整することができる。
酸素透過層(I-b)の全光線透過率は、酸素透過層(I-a)と同様の方法で測定できる。
酸素透過層(I-b)の全光線透過率は、例えば、酸素透過層(I-b)の厚さ、又は酸素透過層(I-b)を構成する材料等を調整することにより、調整することができる。
酸素透過層(I)の水接触角は、例えば、酸素透過層(I)を構成する材料、酸素透過層(I)の表面改質処理条件等を調整することにより、調整することができる。
酸素透過層(I-b)を構成する材料は、要件(6)を満たす酸素透過層(I-b)を形成可能なものであれば特に制限されない。
酸素透過層(I)を構成する材料は、1種単独で用いてもよく、2種以上を組み合わせて用いてもよい。
酸素透過層(I)を構成する材料は、成形加工性、透明性、形状安定性、軽量性、薬剤低収着性、自家蛍光、放射線耐性などの観点から、適切な材料を選択できる。このような観点から、ポリオレフィン系樹脂又はフッ素系樹脂、より好ましくはポリオレフィン系樹脂を選択することにより、本発明の一態様である培養容器は、細胞等を効率よく培養し、細胞等の形態を観察しやすく、また創薬スクリーニング用途や診断用途で使用しやすくなる。
本発明の一態様においては、4-メチル-1-ペンテン単独重合体、及び4-メチル-1-ペンテンと他のモノマーとの共重合体を総称して「4-メチル-1-ペンテン重合体(X)」と称する。
また、4-メチル-1-ペンテン重合体(X)が、4-メチル-1-ペンテンと、エチレン及び炭素数3~20のα-オレフィン(4-メチル-1-ペンテンを除く)から選ばれる少なくとも1種のオレフィンとの共重合体である場合は、その共重合体におけるエチレン及び炭素数3~20のα-オレフィン(4-メチル-1-ペンテンを除く)から選ばれる少なくとも1種のオレフィンから導かれる構成単位の含有量は、好ましくは0~40モル%、より好ましくは0.5~20モル%、さらに好ましくは2~15モル%である。なお、これら構成単位の含有量は、4-メチル-1-ペンテン重合体(X)中の全繰返し構成単位量を100モル%とする。構成単位の含有量が上記範囲内にあると、加工性に優れ均質な培養面が得られ、またフィルムの靭性と強度のバランスが良いため、撓みも少なくなる。
前記4-メチル-1-ペンテン重合体(X)を製造する方法は、4-メチル-1-ペンテン、オレフィン、その他のモノマーを重合させられれば、いずれの方法であってもよい。また、分子量や分子量分布を制御するために連鎖移動剤、例えば水素を共存させてもよい。製造に用いる機器も制限されない。重合法は公知の方法でもよく、気相法、スラリー法、溶液法、バルク法であってもよい。好ましくはスラリー法、溶液法である。また、重合法は単段重合法、又は二段等の多段重合法で、分子量の異なる複数の重合体を重合系中にブレンドする方法であってもよい。単段、多段重合法の何れであっても、連鎖移動剤として水素を用いる場合には、一括投入しても、分割投入、例えば重合初期、中期、終期に投入してもよい。重合は常温で行ってもよく、必要に応じて加温してもよいが、重合の効率の観点から、20℃~80℃で行うことが好ましく、40℃~60℃で行うことがより好ましい。製造に用いる触媒も制限されないが、重合の効率の観点から、例えば国際公開公報2006/054613に記載される固体状チタン触媒成分(I)や、国際公開公報2014/050817に記載される遷移金属化合物(A)を含有するオレフィン重合用触媒(メタロセン触媒)を用いることが好ましい。
4-メチル-1-ペンテン重合体(X)以外の成分としては、耐熱安定化剤、耐光安定化剤、加工助剤、可塑剤、酸化防止剤、滑剤、消泡剤、アンチブロック剤、着色剤、改質剤、抗菌剤、抗黴剤、防曇剤などの添加剤が挙げられる。
また、酸素透過層(I)が多層構造である場合、各層の構成材料及び厚さは同一であってもよく、異なっていてもよい。
酸素透過層(I-b)の厚さは、要件(6)を満たす限り、特に制限されないが、好ましくは20~500μm、より好ましくは25~500μm、さらに好ましくは30~200μmである。
なお、厚さとは、酸素透過層(I)が多層構造である場合には、各層の厚さの合計をいうものである。
以下で説明する、ガスバリア層(II-a)及びガスバリア層(II-b)を総称して、ガスバリア層(II)ともいう。
ガスバリア層(II-a)は、下記要件(3)及び(4)を満たす層である。
(3)温度23℃、湿度0%の時の酸素透過度P(II-a)が3000cm3/(m2×24h×atm)以下である
(4)JIS K 7361-1に準拠して測定した全光線透過率が70%以上である
酸素透過度P(II-a)は、酸素透過層(I-a)と同様の方法で測定することができる。
ガスバリア層(II-a)の酸素透過度P(II-a)は、好ましくは、1cm3/(m2×24h×atm)より大きく3000cm3/(m2×24h×atm)以下、より好ましくは1cm3/(m2×24h×atm)より大きく1000cm3/(m2×24h×atm)以下、さらに好ましくは1cm3/(m2×24h×atm)より大きく500cm3/(m2×24h×atm)以下、特に好ましくは1cm3/(m2×24h×atm)より大きく300cm3/(m2×24h×atm)以下である。
酸素透過度P(II-a)が前記範囲にあるということは、ガスバリア層(II-a)はガスバリア性に優れるといえる。
ガスバリア層(II-a)の酸素透過度P(II-a)は、例えば、ガスバリア層(II-a)の厚さ、又はガスバリア層(II-a)を構成する材料等を調整することにより、調整することができる。
全光線透過率は、酸素透過層(I-a)と同様の方法で測定することができる。
ガスバリア層(II-a)の全光線透過率は、好ましくは85.0%以上、より好ましくは90.0%以上である。全光線透過率の上限は特にないが、通常99.9%である。全光線透過率が前記範囲にあるということは、ガスバリア層(II-a)が透明性に優れるといえる。
ガスバリア層(II-a)の全光線透過率は、例えば、ガスバリア層(II-a)の厚さ、又はガスバリア層(II-a)を構成する材料等を調整することにより、調整することができる。
ガスバリア層(II-b)は、下記要件(5)を満たす層である。
温度23℃、湿度0%の時の酸素透過度P(II-b)が3000cm3/(m2×24h×atm)以下である
酸素透過度P(II-b)は、酸素透過度P(I-a)と同様の方法及び好適態様で測定することができる。
ガスバリア層(II-b)の酸素透過度P(II-b)の好適な範囲及び調整方法は、ガスバリア層(II-a)と同様である。
ガスバリア層(II-b)の全光線透過率は、酸素透過層(I-a)と同様の方法で測定することができる。
ガスバリア層(II-b)の全光線透過率は、好ましくは70%以上、より好ましくは85.0%以上、さらに好ましくは90.0%以上である。全光線透過率の上限は特にないが、通常99.9%である。全光線透過率が前記範囲にあるということは、ガスバリア層(II-b)が透明性に優れるといえる。
ガスバリア層(II-b)の全光線透過率は、例えば、ガスバリア層(II-b)の厚さ、又はガスバリア層(II-b)を構成する材料等を調整することにより、調整することができる。
ガスバリア層(II-a)を構成する材料は、1種単独で用いてもよく、2種以上を組み合わせて用いてもよい。
ガスバリア層(II-b)を構成する材料は、例えば、ポリエチレンテレフタレート(PET)、ナイロン、エチレン-ビニルアルコールコポリマー(EVOH)、ポリグリコール酸、芳香族ポリアミド、ポリ塩化ビニリデン(PVDC)、ポリオレフィンを挙げることができ、これらの中から成形加工性、透明性、形状安定性、軽量性、薬剤低収着性、自家蛍光、放射線耐性などの観点から、適切な材料を選択できる。このような観点から、ポリエチレンテレフタレートまたはポリオレフィンを選択することにより、本発明の一態様である培養容器は、細胞等を効率よく培養し、細胞等の形態を観察しやすく、また創薬スクリーニング用途や診断用途で使用しやすくなる。すなわち、ガスバリア層(II-b)は、好ましくはポリエチレンテレフタレート(PET)およびポリオレフィンからなる群から選択される少なくとも1種を含み、より好ましくはポリエチレンテレフタレート(PET)を含む。
ガスバリア層(II-b)を構成する材料は、1種単独で用いてもよく、2種以上を組み合わせて用いてもよい。
ポリエチレンテレフタレート(PET)は、テレフタル酸及びそのエステル形成性誘導体から選ばれる少なくとも1つと、エチレングリコール及びそのエステル形成性誘導体から選ばれる少なくとも1つとを重縮合することによって得られる重合体である。テレフタル酸のエステル形成性誘導体としては、例えば、テレフタル酸ジメチルなどのテレフタル酸のアルキルエステルなどが挙げられる。エチレングリコールのエステル形成性誘導体としては、例えば、エチレングリコール脂肪酸エステルなどの脂肪酸エステルなどが挙げられる。
ポリエチレンテレフタレートの製造方法は、特に制限されず、公知の方法を用いることができる。
ガスバリア層(II-b)を構成する材料として用いることができるポリオレフィンとしては、例えば、エチレン、1-ブテン、1-ヘキセン、4-メチル-1-ペンテン、1-オクテン等のα-オレフィンの単独重合体又は共重合体;高圧法低密度ポリエチレン;直鎖状低密度ポリエチレン(LLDPE);高密度ポリエチレン;ポリプロピレン(プロピレンの単独重合体);プロピレンと炭素数が2以上10以下のα-オレフィンとの共重合体;エチレン/酢酸ビニル共重合体(EVA);アイオノマー樹脂;フッ素含有環状オレフィン重合体等が挙げられる。なかでも、プロピレンの単独重合体又は共重合体が好ましい。すなわち、ガスバリア層(II-b)は、好ましくはプロピレンの単独重合体又は共重合体を含み、より好ましくはポリプロピレン(プロピレンの単独重合体)を含む。
前記プロピレンの共重合体におけるプロピレンに由来する構成単位以外の構成単位は、特に制限されないが、エチレン及び炭素数4~20のα-オレフィンから選ばれる少なくとも1種のオレフィンとすることができる。前記オレフィンとしては、例えば、エチレン、1-ブテン、1-ヘキセン、1-ヘプテン、1-オクテン、1-デセン、1-テトラデセン、1-ヘキサデセン、1-ヘプタデセン、1-オクタデセン、1-エイコセンが挙げられる。前記オレフィンは、ガスバリア層(II-b)に必要な物性に応じて適宜選択することができる。
ポリエチレンテレフタレートおよびポリオレフィン以外の成分としては、耐熱安定化剤、耐光安定化剤、加工助剤、可塑剤、酸化防止剤、滑剤、消泡剤、アンチブロック剤、着色剤、改質剤、抗菌剤、抗黴剤、防曇剤、接着剤などの添加剤が挙げられる。
また、ガスバリア層(II)が多層構造である場合、各層の構成材料及び厚さは同一であってもよく、異なっていてもよい。
(7)ポリエチレンテレフタレート(PET)およびポリオレフィンからなる群から選択される少なくとも1種と、粘着剤との混合物を含む
(8)ポリエチレンテレフタレート(PET)およびポリオレフィンからなる群から選択される少なくとも1種を含む層と、粘着剤を含む層と、を有する
ポリエチレンテレフタレート(PET)およびポリオレフィンからなる群から選択される少なくとも1種と、粘着剤との混合物を含むとは、ガスバリア層(II-b)の1層が、ポリエチレンテレフタレート(PET)およびポリオレフィンからなる群から選択される少なくとも1種と、粘着剤との組成物から構成されることを意味する。
該組成物中におけるポリエチレンテレフタレート(PET)およびポリオレフィンからなる群から選択される少なくとも1種の含有量は特に制限されない。
該組成物中における粘着剤の含有量は特に制限されない。
ポリエチレンテレフタレート(PET)およびポリオレフィンからなる群から選択される少なくとも1種と、粘着剤との質量比は、特に制限されない。
粘着剤については後述する。
ポリエチレンテレフタレート(PET)およびポリオレフィンからなる群から選択される少なくとも1種を含む層と、粘着剤を含む層と、を有するとは、ガスバリア層(II-b)が、ポリエチレンテレフタレート(PET)およびポリオレフィンからなる群から選択される少なくとも1種を含む層と、粘着剤を含む層との少なくとも2層を有することを意味する。
ポリエチレンテレフタレート(PET)およびポリオレフィンからなる群から選択される少なくとも1種を含む層の厚さは特に制限されないが、好ましくは10~500μm、より好ましくは50~100μmである。
粘着剤を含む層の厚さは特に制限されないが、好ましくは1~100μm、より好ましくは5~20μmである。
ポリエチレンテレフタレート(PET)およびポリオレフィンからなる群から選択される少なくとも1種を含む層と、粘着剤を含む層の厚さの比は特に制限されないが、好ましくは500:1~10:100、より好ましくは100:5~50:20である。
粘着剤については後述する。
培養容器(α)は、培養容器の培養面の少なくとも一部が、要件(1)及び(2)を満たす酸素透過層(I-a)と、要件(3)及び(4)を満たすガスバリア層(II-a)との積層領域を有する。
培養面の一部の領域のみが酸素透過層(I-a)とガスバリア層(II-a)との積層領域である場合、培養面の前記積層領域以外の領域は、酸素透過層(I-a)で構成される領域を有することが好ましく、培養面の前記積層領域以外の全ての領域は、酸素透過層(I-a)で構成される領域であることがより好ましい。
培養容器(α)の培養面の、酸素透過層(I-a)とガスバリア層(II-a)との積層領域以外の部材を構成する材料も特に制限されず、公知の材料を用いることができる。かかる材料としては、例えば、ポリスチレン(PS)、ポリジメチルシロキサン(PDMS)、熱硬化性樹脂、環状オレフィンポリマー、環状オレフィンコポリマー、ガラス等が挙げられる。培養容器(α)の培養面の、酸素透過層(I-a)とガスバリア層(II-a)と積層領域以外の部材を構成する材料は、酸素透過層(I-a)を構成する材料と同じであることが好ましい。
培養容器(β)は、前記底面部材の少なくとも一部が、少なくとも酸素透過層(I-b)と、ガスバリア層(II-b)とを有し、前記ガスバリア層(II-b)が前記酸素透過層(I-b)の下に設けられている。
底面部材の一部の領域のみが、酸素透過層(I-b)とガスバリア層(II-b)とを有する領域である場合、底面部材における、該両方の層を有する領域以外の領域は、酸素透過層(b)で構成される領域であることが好ましく、底面部材における該両方の層を有する領域以外の全ての領域は、酸素透過層(I-b)で構成される領域であることがより好ましい。
培養容器(β)は、培養面全体が酸素透過層(I-b)とガスバリア層(II-b)とを有する領域であることが好ましい。
ガスバリア層(II-b)は、酸素透過層(I-b)の下に直接重ねられてもよいし、酸素透過層(I-b)とガスバリア層(II-b)との間に、他の層(例えば金属蒸着層、上記接着層など)が含まれていてもよい。該他の層は、培養容器(β)の内部側から、酸素透過層(I-b)、ガスバリア層(II-b)、他の層の順で重ねられていてもよい。
培養容器(β)の底面部材の、酸素透過層(I-b)とガスバリア層(II-b)とを有する領域以外の領域を構成する材料も特に制限されず、公知の材料を用いることができる。かかる材料としては、例えば、ポリスチレン(PS)、ポリジメチルシロキサン(PDMS)、熱硬化性樹脂、環状オレフィンポリマー、環状オレフィンコポリマー、ガラス等が挙げられる。培養容器(β)の底面部材の、酸素透過層(I-b)とガスバリア層(II-b)とを有する領域以外の領域を構成する材料は、酸素透過層(I-b)を構成する材料と同じであることが好ましい。
培養容器(β)の底面部材の、酸素透過層(I-b)とガスバリア層(II-b)とを有する領域の全光線透過率は、例えば、酸素透過層(I-b)とガスバリア層(II-b)の厚さ、又は酸素透過層(I-b)とガスバリア層(II-b)を構成する材料等を調整することにより、調整することができる。
前記接着層を構成する接着材料は、所望の接着性を有する接着層を形成可能なものであれば特に制限されないが、細胞培養、医療用途等で用いられる器具の接着層の形成に一般的に用いられるものを用いることができる。
また、前記接着材料は、例えば、従来から知られたシーラント樹脂を使用できる。前記シーラント樹脂は、例えば、低密度ポリエチレン(LDPE)、直鎖状低密度ポリエチレン(LLDPE)、高密度ポリエチレン(HDPE)等のポリエチレン、酸変性ポリエチレン、ポリプロピレン(PP)、酸変性ポリプロピレン、プロピレンの共重合体、エチレン-ビニルアセテート共重合体、ポリプロピレン-ビニルアセテート共重合体、エチレン-(メタ)アクリル酸エステル共重合体、エチレン-(メタ)アクリル酸共重合体及びそれらの金属架橋物であるアイオノマー等のポリオレフィン系樹脂、ポリ塩化ビニル系樹脂、シリコーン樹脂等を挙げることができ、なかでも低温シール性の観点からポリオレフィン系樹脂が好ましく、安価であることからポリエチレンが特に好ましい。
前記接着材料は、1種単独で用いてもよく、2種以上を組み合わせて用いてもよい。
前記接着層の厚さは、0.1μm~200μmの範囲内とすることができ、好ましくは5μm~100μm、より好ましくは10μm~60μm、特に好ましくは15μm~40μmの範囲内である。この範囲内であると、接着層を加工性やヒートシール性に優れたものとすることができる。
培養容器(α)又は培養容器(β)の製造方法は、特に制限されず、製造に用いる機器も制限されない。
培養容器(α)の全ての部材が酸素透過層(I-a)を構成する材料から形成される場合には、例えば、酸素透過層(I-a)を構成する材料を含むフィルム又はシートを形成し、必要に応じてそのフィルム又はシートを成形して所望の形状とした後、ガスバリア層(II-a)を積層して、培養容器(α)を作製することができる。また、培養容器(α)は、酸素透過層(I-a)を構成する材料を、押出成形、溶液キャスト成形、射出成形、ブロー成形等の方法により、直接成形した後、ガスバリア層(II-a)を積層することによっても得られる。
培養容器(β)の製造方法は、側壁部材に、底面部材を貼付する工程を含んでもよい。例えば、培養容器(β)は、側壁部材に、酸素透過層(I-b)を構成する材料を含むフィルム又はシートを貼付することで底面部材と側壁部材とを接合した後、該フィルム又はシートの下に、ガスバリア層(II-b)を構成する材料を含むフィルム又はシートをさらに貼付することで製造することができる。また、培養容器(β)は、酸素透過層(I-b)を構成する材料を含むフィルム又はシートの下に、予め、ガスバリア層(II-b)を構成する材料を含むフィルム又はシートを貼付しておき、該貼付された2つのフィルム又はシートを側壁部材に貼付することで底面部材と側壁部材とを接合して製造することができる。
培養中とは、細胞等を培養する期間中の任意のタイミングとの意味である。
着脱可能とは、培養容器の使用者の通常の力で、取り付け及び取外しができるとの意味である。
ガスバリア層(II-b)の領域の一部のみを酸素透過層(I-b)から取り外しできる態様としては、例えば、培養容器が少なくとも1つのウェルを有するプレートである場合、各ウェルに対応する格子状の切れ目を予めガスバリア層(II-b)に入れておき、細胞に十分な酸素を供給したいウェルに対応するガスバリア層(II-b)のみを取り外すことが挙げられる。また、培養容器が少なくとも1つのウェルを有するプレートである場合、ガスバリア層(II-b)の領域の一部(例えば、1/4、1/3、1/2)、すなわち、プレートが有するウェルのうちの一部(例えば、1/4、1/3、1/2)の数のウェルに対応する領域だけを取り外して、該一部の数のウェルにおいて酸素透過層(I-b)を露出させる態様、ガスバリア層(II-b)の各ウェルに対応する領域の一部(例えば、1/4、1/3、1/2)だけを取り外して、該各ウェルの酸素透過層(I-b)の領域の一部(例えば、1/4、1/3、1/2)を露出させる態様も挙げられる。
[剥離強度]
ガスバリア層(II-b)の酸素透過層(I-b)に対する剥離強度は、0.01~0.20N/25mmであることが好ましく、より好ましくは0.02~0.15N/25mmである。剥離強度が上記下限値以上であると、意図しない剥離、例えば、ガスバリア層(II-b)が自重で酸素透過層(I-b)から剥離してしまうことを防ぐことができる。一方、剥離強度が上記上限値以下であると、剥離時の衝撃による細胞等への損傷、例えば、剥離音による振動又は培養容器を持つ手の振動により生じる細胞等への損傷を防ぐことができる。
ガスバリア層(II-b)から作製した、幅25mmの試験片の剥離強度を測定する面を下向きにして、酸素透過層(I-b)の上面に貼付し、2kgローラーを1往復させる。これを23℃、RH50%の環境下に30分間保持した後、剥離試験機を用い、JIS Z 0237に準拠して、23℃、RH50%の環境下、剥離角度180°、引張速度10mm/分の条件にて、剥離強度[N/25mm]を測定する。剥離試験機としては、例えば、株式会社東洋精機製作所製、ストログラフE-Sを用いることができる。試験を3回行い、その平均値を算出する。
本発明の一態様である、細胞、組織、又は器官の培養方法は、
培養容器(α)を用いて、細胞、組織、又は器官を培養する工程(A-a)、
前記ガスバリア層(II-a)を前記酸素透過層(I-a)から取り外す工程(B-a)、及び
前記工程(B-a)で得られた培養容器を用いて、細胞、組織、又は器官をさらに培養する工程(C-a)を含む。
培養容器(β)を用いて、細胞、組織、又は器官を培養する工程(A-b)、
前記ガスバリア層(II-b)を前記酸素透過層(I-b)から取り外す工程(B-b)、及び
前記工程(B-b)で得られた培養容器を用いて、細胞、組織、又は器官をさらに培養する工程(C-b)を含む。
培養容器(α)又は培養容器(β)は、前記酸素透過層(I)の水接触角が、30~120°であることが好ましい。
培養容器(α)又は培養容器(β)は、前記酸素透過層(I)が、4-メチル-1-ペンテン重合体(X)を含むことが好ましい。
培養容器(α)又は培養容器(β)は、培養面、好ましくは培養面の培地及び/又は細胞等が接触する側に、天然高分子材料、合成高分子材料、又は無機材料がコーティングされていることが好ましい。
培養容器(α)又は培養容器(β)は、前記ガスバリア層(II)が、ポリエチレンテレフタレート(PET)を含むことが好ましい。
培養に用いる培地は、細胞等が増殖できる培地であれば特に制限されず、使用細胞種に合わせて適宜選択すればよい。
培養に用いる培地の量及び培地交換の頻度は特に制限されない。培養温度は特に制限されないが、通常は25~40℃程度で行う。
工程(A-a)又は工程(A-b)では、酸素供給の少ない状態で初期の培養を行うので、接着性がより良好になりやすい。
培養容器(α)は、ガスバリア層(II)を有するので、容器内部への酸素透過が抑制されたものであるが、ガスバリア層(II)を酸素透過層(I)から取り外すことにより酸素透過層が露出されるので、培養容器(α)又は培養容器(β)は、容器内部へ充分に酸素が透過するものとなる。
細胞等を培地中で培養する方法は特に制限されず、工程(A-a)又は工程(A-b)に準じて行うことができる。培養に用いる培地、培養に用いる培地の量及び培地交換の頻度は、工程(A-a)又は工程(A-b)と同じであってもよいし、異なってもよい。
工程(C-a)又は工程(C-b)では、充分に酸素供給される状態で細胞等を培養するので、細胞等の機能が正常に維持されやすい。
[材料]
TPXフィルム:4-メチル-1-ペンテン重合体フィルム(厚さ50μm、三井化学東セロ株式会社製、商品名:オピュランX-88B)
PETフィルム1:PETと粘着剤の混合物からなる市販のPETフィルム(厚さ80μm、ビーエム機器株式会社製、商品名:qPCRプレートシール(ABI、圧着タイプ)
PETフィルム2:基材層(PET層、厚さ75μm)、及び、粘着剤層(厚さ10μm)からなる市販のPETフィルム(日榮新化株式会社、商品名:PET75-H270(10))
PPフィルム:基材層(PET層、厚さ60μm)、及び粘着剤層(厚さ10μm)からなる市販のPPフィルム(日榮新化株式会社、商品名:PP60-H270(10))
PDMS容器:底面が、厚さ350μmのPDMS(ジメチルポリシロキサン)フィルムで構成される市販の96ウェル培養容器(製品名G-plate、VECELL社製型番V96WGPB-10)
PS容器:PS(ポリスチレン)製の底面の厚さが1000μmである市販の24ウェルPS培養容器(コーニング社製)
上述のTPXフィルム、PETフィルム1~2、PPフィルム、PDMS容器及びPS容器を測定サンプルとして、以下で詳述する方法により、酸素透過度、及び全光線透過率を測定した。結果を表1に示す。
測定サンプルについて、株式会社東洋精機製作所製差圧式ガス透過率測定装置MT-C3を用いて温度23℃、湿度0%の環境下にて酸素透過係数を測定した。測定部径は70mm(透過面積は38.46cm2)とした。酸素透過係数が大きいことが予想されたため、予めサンプルにアルミニウムマスクを施し、実透過面積を5.0cm2とした。
測定した酸素透過係数[cm3×mm/(m2×24h×atm)]の値をフィルム(又は培養容器底面)の厚さ(μm)で除して、酸素透過度[cm3/(m2×24h×atm)]を算出した。
測定サンプルについて、ヘイズメーター(NDH2000、日本電色工業社製)を用い、JIS K 7361-1に準拠して全光線透過率を測定した。
水接触角の測定は、日本工業規格JIS-R3257(基板ガラス表面のぬれ性試験方法)に準じて行った。25±5℃、50±10%の恒温恒湿条件下で水滴の形状を球形とみなせる4μL以下の容量の水滴を、測定サンプルの表面に滴下し、静滴法により、測定サンプル表面に水滴が接触した直後から1分以内の測定サンプルと水滴の接触界面の角度を測定した。
0.1Mの塩酸溶液(容量分析用、富士フイルム和光純薬株式会社製)を注射用水(日本薬局方、大塚製薬株式会社製)で100倍希釈し、0.001Mの塩酸溶液を調製してろ過滅菌をした。3mg/mLのコラーゲン溶液(セルマトリックスTypeI-P、ブタ腱由来、新田ゼラチン株式会社製)を0.001Mの塩酸溶液で6倍希釈し、0.5mg/mLのコラーゲン溶液を調製した。
ラット初代凍結肝細胞(Hepatocyte)を含む細胞懸濁液を入れた遠沈管(50mL)に培地(A)を加えた。培地(A)は、ウシ胎児血清(Fetal Bovine Serum、FBS、富士フイルム和光純薬株式会社製)を1.5mL、注射用水(扶桑薬品工業株式会社製)で3.0g/mLに希釈したL-プロリン(培養用、富士フイルム和光純薬株式会社製)溶液を0.15mL、エタノール(分子生物学用、富士フイルム和光純薬株式会社製)で1×10-3Mに希釈したデキサメタゾン(生化学用、富士フイルム和光純薬株式会社製)溶液を1.5μL、エタノールで36mMに希釈したハイドロコルチゾン(培養用、富士フイルム和光純薬株式会社製)溶液を21μL、注射用水で1.0mg/mLに希釈したBSA溶液を用いて、さらに20μg/mLに希釈した上皮成長因子(Epidermal growth factor、EGF、細胞生物学用、富士フイルム和光純薬株式会社製)溶液を15μL、インスリン溶液(10mg/mL in HEPESS、シグマアルドリッチジャパン合同会社製)を8.7μL、ペニシリン-ストレプトマイシン溶液(5000units/mLペニシリン、5000μg/mLストレプトマイシン含有、培養用、富士フイルム和光純薬株式会社製)を0.3mL、D-MEM培地(4500mg/mL D-グルコース、584mg/mL L-グルタミン、15mg/mLフェノールレッド、110mg/mLピルビン酸ナトリウム、3700mg/mL炭酸水素ナトリウム含有、培養用、富士フイルム和光純薬株式会社製)を13mL加えて調製した。細胞密度の調整は、ラット初代凍結肝細胞を含む細胞懸濁液の細胞数を調整する方法で実施し、1.0×105cells/cm2の細胞密度で培養した。
細胞播種の24時間後、培養容器の培地を除去した後、新たに培地(A)を0.5mL添加し、蛍光式の酸素センサー(FireSting酸素モニター、株式会社ビー エー エス社製)を用いて培地中の酸素分圧(hPa)を測定した。測定は、加湿インキュベーター中で実施し、センサーをジャッキで培養容器底面から80μmの高さに設置し、1時間測定した。測定開始から1時間後の培地中の酸素分圧(hPa)を、1013hPa(大気1気圧)で除して100をかけた値(%)を算出し、培地中の酸素濃度とした。代表的な3ウェルで測定し、平均値を算出した。
細胞播種24時間後、又は48時間後の培養容器中の培地を除去した後、培地で希釈したLuciferin-CEEを添加して、さらに3時間培養した。培養後の細胞をLuciferin-CEEを含む培地を同伴して96ウェルプレートに移した後、Luciferin Detection RegentとReconstitution Bufferの混合液を添加して、室温で遮光して1時間反応させた。1時間後、ルミノメーターで発光量(Relative Light Unit、RLU)を測定した。
蛋白量は、培地で希釈したLuciferin-CEE溶液を除去後、PBS(-)を培地200μL添加した後、セルスクレーパーを用いてエッペンチューブに細胞を回収し、遠心した(4℃、22000×g、10分間)。その後、上澄みを除去し、0.1M水酸化ナトリウム溶液を100μL添加した後、PierceTMBCA Protein Assay Kit(Thermo Fisher Scientific社製)を使用して蛋白量を測定した。波長450nmの吸光度をプレートリーダー(SPECTRA max PLUS384、Molecular Devices社製)で測定した。
ルミノメーターで得られたLuciferin-CEE溶液の代謝活性量(pmol/L)をP450-GloTM CYP1A1 Assay kit(Promega社製)を使用して測定し、吸光度から得られたタンパク量及びLuciferin-CEE溶液の反応時間で除することにより、代謝活性値(pmol/min/mg protein)を算出した。代表的な3ウェルで測定し、平均値を算出した。
代謝活性維持率は以下の式により算出した。
代謝活性維持率(%)=播種48時間後の代謝活性値/播種24時間後の代謝活性値×100
ヒト初代凍結肝細胞(Hepatocyte)を含む細胞懸濁液を入れた遠沈管(50mL)に培地(B)を加えた。培地(B)は、ウシ胎児血清(Fetal Bovine Serum、FBS、富士フイルム和光純薬株式会社製)を2.5mL、Antibiotic-Antimycotic(10000units/mLペニシリン、10000μg/mLストレプトマイシン、25μg/mLアンホテリシンB含有、GibcoTM)を2.5mL、エタノール(分子生物学用、富士フイルム和光純薬株式会社製)で1×10-3Mに希釈したデキサメタゾン(生化学用、富士フイルム和光純薬株式会社製)溶液を0.05mL、インスリン溶液(10mg/mL in HEPESS、シグマアルドリッチジャパン合同会社製)を0.02mL、GlutaMAX溶液(GibcoTM)を0.5mL、HEPES buffer solution(生化学用緩衝剤、同仁化学株式会社製)を0.75mL、注射用水で100mMに調製したL-アスコルビン酸2-リン酸エステル三ナトリウム(富士フイルム和光純薬株式会社製)を0.05mL、注射用水で1.0mg/mLに希釈したBSA溶液を用いて、さらに20μg/mLに希釈した上皮成長因子(Epidermal growth factor、EGF、細胞生物学用、富士フイルム和光純薬株式会社製)溶液を0.5mL、William’s E Medium(2000mg/mL D-グルコース、10mg/mLフェノールレッド、25mg/mLピルビン酸ナトリウム、2200mg/mL炭酸水素ナトリウム含有、培養用、Giboco)を43.13mL加えて調製した。細胞密度の調整は、ラット初代凍結肝細胞を含む細胞懸濁液の細胞数を調整する方法と同様に実施し、1.0×105cells/cm2の細胞密度で培養した。
培養容器にヒト初代凍結肝細胞の細胞懸濁液を0.5mL添加し、細胞密度が1×105cells/cm2となるように播種し、37℃、5%CO2下でインキュベートし、24時間培養後の状態を顕微鏡下で観察し、以下の基準で評価した。また、顕微鏡下で写真撮影を行った。
AA:肝細胞が培養面に接着し、伸展している状態が観察される。
BB:肝細胞が培養面に接着しているが丸くなり伸展していないか、もしくは一部に剥離した状態が観察される。
培養容器底面を構成するフィルムに接着させたフィルム(PETフィルム1、2、PPフィルムのいずれか)をフィルム縁部から剥離した。培養容器底面を構成するフィルム及び剥離したフィルム(PETフィルム1、2、PPフィルムのいずれか)の破れの有無等を確認し、以下基準で評価した。
AA:培養容器底面を構成するフィルム及び剥離したフィルム(PETフィルム1、2、PPフィルムのいずれか)の破れがなく、培養容器底面を構成するフィルムの撓みも確認されず、剥離性が良好であった。
BB:培養容器底面を構成するフィルム及び/又は剥離したフィルム(PETフィルム1、2、PPフィルムのいずれか)の破れがあるか、培養容器底面を構成するフィルムが撓み、剥離性が不良であった。
25mm×150mmに切り取ったフィルム(PETフィルム1、2、PPフィルムのいずれか)の剥離紙を剥離して、剥離した面を、50mm×150mmに切り取ったTPXフィルム、又はPDMSフィルム上面に貼付し、2kgローラーを1往復させた。これを23℃、RH50%の環境下に30分間保持した後、剥離試験機(株式会社東洋精機製作所製、ストログラフE-S)を用い、JIS Z 0237に準拠して、23℃、RH50%の環境下、剥離角度180°、引張速度10mm/分の条件にて、剥離強度[N/25mm]を測定した。試験は3回行い、その平均値を算出した。
TPXフィルムに対し、常圧プラズマ表面処理装置(積水化学工業製)を用いて、チャンバー内を窒素の気流で満たし、プラズマ処理した(処理速度2m/min、出力4.5kW、2往復)。
前記プラズマ処理したTPXフィルムを8cm×12cmサイズにカットし、ウェル底無しPS製24ウェル容器枠に、医療用粘着剤(スリーエム製)を介して密着させ、培養プレートの底面が、TPXフィルムで構成される24ウェル培養プレートを作製した。
上記培養プレート底面のTPXフィルムに対して、容器枠とは逆側に、PETフィルム1を圧着ゴムローラー(ローラー幅:45mm、ローラー質量2kg)を2往復させ、一定圧で圧着して、培養プレートの底面が、TPXフィルムとPETフィルム1との積層体で構成される24ウェル培養プレートを作製した。すなわち、PETフィルム1の剥離紙を剥離した面を、培養プレート底面を構成するTPXフィルムの下面に接着した。
その後、上記24ウェル培養プレートを耐ガンマ線袋に梱包して10kGyのガンマ線を照射し滅菌した。
細胞播種から24時間後、インキュベーターから培養容器A~Dを取り出した。
培養容器(1)A、Bは、底面のPETフィルムを剥離した後、インキュベーターに戻して、さらに24時間培養を行った。
培養容器(1)Cでは、培地中の溶存酸素濃度を測定した。
培養容器(1)Dでは、肝細胞の正常な機能の指標として、代謝活性値を測定した。
細胞播種から48時間後、PETフィルム1を剥離した培養容器(1)Aをインキュベーターから取り出し、溶存酸素濃度の測定を行った。
細胞播種から48時間後、PETフィルム1を剥離した培養容器(1)Bをインキュベーターから取り出し、代謝活性値の測定を行った。
結果を表1に示す。
播種24時間後に培養容器(1)Eをインキュベーターから取り出し、底面のPETフィルム1を剥離して、剥離性の評価及び細胞接着性の評価を行った。結果を表1及び図2に示す。
培養容器(1)の底面のPETフィルム1をPPフィルムに変更したこと、及び、TPXフィルムへの表面処理をテーブル型コロナ処理装置(春日電機製)を用いたコロナ処理(処理速度3m/min、出力0.5kW、2往復)に変更したこと以外は、実施例1と同様にしてラット凍結肝細胞およびヒト凍結肝細胞の培養及び評価を実施したものを実施例2とした。結果を表1に示す。
培養容器(1)の底面のPETフィルム1をPETフィルム2に変更したこと以外は、実施例1と同様にしてラット凍結肝細胞およびヒト凍結肝細胞の培養及び評価を実施したものを実施例3とした。結果を表1に示す。
PDMS容器の底面を構成するPDMSフィルムに対して、PETフィルム2を圧着ゴムローラー(ローラー幅:45mm、ローラー質量2kg)を2往復させ、一定圧で圧着して、培養プレートの底面が、PDMSフィルムとPETフィルム2との積層体で構成される96ウェル培養プレートを作製し、各ウェルに、0.5mg/mLのコラーゲン溶液を0.1mL添加した後、余分なコラーゲン溶液を除去した。室温で30~60分間静置した後、ダルベッコPBS(-)で洗浄して、一晩、室温で乾燥させた。実施例1と同様にしてラット凍結肝細胞およびヒト凍結肝細胞の培養及び評価を実施したものを実施例4とした。結果を表1に示す。
培養容器(1)の底面のPETフィルム1を剥離しなかったこと以外は、実施例1と同様にしてラット凍結肝細胞の培養及び評価を実施したものを参考例1とした。結果を表2に示す。ヒト凍結肝細胞の培養及び評価は行わなかった。
比較例1では、培養プレートの底面がTPXフィルムとPETフィルム1との積層体で構成される24ウェル培養プレートの代わりに、市販の24ウェルPS培養容器(PS)を用いたこと以外は実施例1と同様にしてラット凍結肝細胞の培養及び評価を実施した。結果を表2に示す。ヒト凍結肝細胞の培養及び評価は行わなかった。
比較例2では、培養プレートの底面が、TPXフィルムで構成される24ウェル培養プレートを用い、PETフィルム1を貼付しなかったこと以外は実施例1と同様にしてラット凍結肝細胞及びヒト凍結肝細胞の培養及び評価を実施した。結果を表2及び図3に示す。
実施例1~4はいずれも、播種24時間後における細胞接着性が良好であった。また、代謝活性維持率も良好で、播種24時間後から播種48時間後にかけて起こる、細胞の代謝活性の低下を抑制できた。実施例1~4の培養容器は、肝細胞を良好に接着させ、肝細胞の正常な機能を維持できるものであることが明らかになった。
この出願は、2021年11月9日に日本国特許庁に出願された特願2021-182633号を基礎とする優先権を主張し、その開示の全てをここに取り込む。
Claims (13)
- 細胞、組織、又は器官を培養する培養容器であって、
前記培養容器が、底面部材と、側壁部材とを有し、
前記底面部材と前記側壁部材とが接合することで少なくとも一つの培養空間が形成され、
前記底面部材の少なくとも一部が、少なくとも酸素透過層(I-b)と、ガスバリア層(II-b)とを有し、
前記ガスバリア層(II-b)が、前記酸素透過層(I-b)の下に、前記酸素透過層(I-b)から着脱可能に重ねられ、
前記ガスバリア層(II-b)が、下記要件(5)を満たし、
前記酸素透過層(I-b)が、下記要件(6)を満たす、
培養容器。
(5)温度23℃、湿度0%の時の酸素透過度P(II-b)が3000cm3/(m2×24h×atm)以下である
(6)温度23℃、湿度0%の時の酸素透過度P(I-b)が前記P(II-b)の6.0倍以上である - 前記ガスバリア層(II-b)が、ポリエチレンテレフタレート(PET)およびポリオレフィンからなる群から選択される少なくとも1種を含み、かつ、下記要件(7)または(8)を満たす、請求項1に記載の培養容器。
(7)ポリエチレンテレフタレート(PET)およびポリオレフィンからなる群から選択される少なくとも1種と、粘着剤との混合物を含む
(8)ポリエチレンテレフタレート(PET)およびポリオレフィンからなる群から選択される少なくとも1種を含む層と、粘着剤を含む層と、を有する - 下記方法で測定される、前記ガスバリア層(II-b)の前記酸素透過層(I-b)に対する剥離強度が、0.01~0.20N/25mmである、請求項1又は2に記載の培養容器。
[幅25mmのガスバリア層(II-b)を酸素透過層(I-b)に貼付した後、剥離試験機を用いて、ガスバリア層(II-b)を酸素透過層(I-b)から剥離角度180°で10mm/分の速度で剥離する試験を行う。] - 前記酸素透過層(I-b)が、4-メチル-1-ペンテン重合体(X)を含む、請求項1又は2に記載の培養容器。
- 前記4-メチル-1-ペンテン重合体(X)が、4-メチル-1-ペンテン単独重合体(x1)並びに、4-メチル-1-ペンテンと、エチレン及び炭素数3~20のα-オレフィン(4-メチル-1-ペンテンを除く)から選ばれる少なくとも1種のオレフィンとの共重合体(x2)から選択される少なくとも1種の重合体である、請求項4に記載の培養容器。
- 前記P(I-b)が、20000~60000cm3/(m2×24h×atm)である、請求項1又は2に記載の培養容器。
- 前記底面部材の、前記酸素透過層(I-b)と前記ガスバリア層(II-b)とを有する領域の、JIS K 7361-1に準拠して測定した全光線透過率が70%以上である、請求項1又は2に記載の培養容器。
- 前記酸素透過層(I-b)の、JIS K 7361-1に準拠して測定した全光線透過率が70%以上である、請求項1又は2に記載の培養容器。
- 前記ガスバリア層(II-b)の、JIS K 7361-1に準拠して測定した全光線透過率が70%以上である、請求項1又は2に記載の培養容器。
- 前記酸素透過層(I-b)の水接触角が、30~120°である、請求項1または2に記載の培養容器。
- 前記底面部材の培養面に、天然高分子材料、合成高分子材料、又は無機材料がコーティングされた、請求項1または2に記載の培養容器。
- 細胞、組織、又は器官の培養方法であって、
請求項1または2に記載の培養容器を用いて、細胞、組織、又は器官を培養する工程(A-b)、
前記ガスバリア層(II-b)を前記酸素透過層(I-b)から取り外す工程(B-b)、及び
前記工程(B-b)で得られた培養容器を用いて、細胞、組織、又は器官をさらに培養する工程(C-b)、
を含む、培養方法。 - 請求項1または2に記載の培養容器の製造方法であって、
前記方法が、
前記側壁部材に、前記底面部材を貼付する工程を含む、培養容器の製造方法。
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