Culture vessel, method for manufacturing the same, and culture method

Abstract

An embodiment of the present invention pertains to a culture vessel in which enough oxygen for the growth of cells or the like is supplied from a bottom section of the culture vessel having cultured matter such as cells adhered thereto, and which has a culture space suitable for three-dimensionally controlling the growth of the cultured matter, and further, from which the grown cultured matter can be removed without damage thereto.　The culture vessel has a culture space for culturing cultured matter. The culture vessel has: a vessel member (B) that has an inverse recessed portion at a lower surface of the bottom section, and that is provided with a first through hole at an end of the inverse recessed portion; and a film member (A) that has oxygen permeability. The film member (A) is detachably attached with the vessel member (B). When the film member (A) is attached to the vessel member (B), the inverse recessed portion and the film member (A) form a space α. The first through hole located at an end of the space α receives the cultured matter. The space α is a part of the culture space.

Description

[Technical Field]

[0001] One aspect of the present invention relates to a culture vessel, a method for producing the same, and a culture method. [Background technology]

[0002] Cells, tissues, or organs (hereinafter also referred to as "cells, etc.") cannot be cultured unless the conditions are suitable for their growth. Therefore, it is necessary to place them in culture vessels such as dishes, plates, or flasks along with a culture medium containing appropriate nutrients, and to maintain the temperature, humidity, and gas concentration of the culture vessel at predetermined levels.

[0003] Furthermore, in order to efficiently achieve the above cultivation, sufficient and appropriate oxygen supply must be provided. Culture vessels made of materials such as glass or polystyrene, which have low gas permeability, require gas supply into the culture vessel. Therefore, an opening is provided at the top of the culture vessel, such as a cap or lid, to ensure gas supply from inside the incubator to the inside of the vessel. However, cells and other organisms usually adhere to the upper surface of the bottom of the culture vessel, or float near the bottom. Generally, the upper surface of the bottom of the culture vessel is covered with culture medium, so the rate of oxygen diffusion in the medium becomes the rate-limiting factor. In particular, the oxygen supply to cells and other organisms at the bottom of the culture vessel is insufficient, and it has long been known that this hinders cell proliferation (Non-Patent Literature 1).

[0004] Traditionally, cells cultured in vitro (outside the body) using culture vessels have been used, for example, to evaluate drug efficacy and safety in order to accelerate drug development.

[0005] Furthermore, cells cultured in vitro using culture vessels are needed in iPS cell drug discovery, where abnormal cells are reproduced using iPS cells to find therapeutic drugs, and in regenerative medicine, where damaged tissues in the body are regenerated through cell transplantation. In other words, there is a demand for advanced culture technology that can reproduce the growth of cells in vivo in vitro (outside the body).

[0006] However, a problem with conventional culture vessels is that cells are cultured two-dimensionally on a flat surface, resulting in a morphology that differs significantly from that of living organisms. Specifically, cells cultured two-dimensionally on a flat surface tend to become flat and elongated. Therefore, development of three-dimensional culture technology that can reproduce the morphology of cells in living organisms is underway.

[0007] For example, there is a known cell culture technique that creates three-dimensional structures as aggregates called spheroids by adhering cells together, such as with a bio-3D printer (Non-Patent Literature 2).

[0008] Technological development is also progressing regarding culture vessels with microfabrication known as microfluidics. For example, a technique for culturing nerve cells is known that uses a cell culture chip to rapidly grow axonal bundles extending from nerve cell bodies in vitro (Patent Document 1). Furthermore, a cell culture vessel has been developed that has a shape suitable for cell culture, and in which the microfluidic section of the culture vessel is detachable, allowing for reuse by replacing the microfluidic section (Patent Document 2).

[0009] Furthermore, a technology has been developed for a culture vessel in which microchannels are controlled three-dimensionally by bonding multiple components together. This technology involves forming channels on the surface of a plastic substrate and bonding a separately manufactured plastic substrate to it with an adhesive to create a microchannel device (Patent Document 3). Since the use of adhesives in microfluidic devices can inhibit cell culture, research is also being conducted on technologies to create microfluidic devices by bonding plastic substrates together using thermocompression without using adhesives (Patent Document 4). [Prior art documents] [Patent Documents]

[0010] [Patent Document 1] International Publication No. 2017 / 187696 [Patent Document 2] International Publication No. 2006 / 123570 [Patent Document 3] Japanese Unexamined Patent Application Publication No. 2002-139419 [Patent Document 4] Japanese Unexamined Patent Application Publication No. 2016-203291 [Non-Patent Document]

[0011] [Non-Patent Document 1] Stevens, K. M., Oxygen requirements for liver cells in vitro., Nature, 206, 199 (1965) [Non-Patent Document 2] Journal of the Japan Society of Printing, Vol. 51, No. 1, pp. 11-17 [Summary of the Invention] [Problems to be Solved by the Invention]

[0012] In the technique of Patent Document 1, by three-dimensionally controlling the culture space with a cell culture chip, it is possible to produce a delicate organoid having a function similar to that of a tissue in vivo and a controlled morphology. However, when removing the cells cultured in a narrow culture space, it is necessary to suck them out with a pipette from the outlet of the microchannel, and there is a concern that the cultured cells may be damaged. In addition, the problem was also that the physical load on the cultured cells was large.

[0013] In the technique of Patent Document 2, although it was an advantageous technique in terms of removing the cultured cells, due to the structure in which the upper side of the flow path was open, it was not suitable for three-dimensionally controlling the growth of cells. Furthermore, the problem was also that the cells during culture adhered to the bottom surface of the culture container and the structure was such that sufficient oxygen could not be supplied from the bottom surface. If sufficient oxygen could not be supplied to the cells during culture, there was a concern that the cells would grow distorted or peel off from the culture container.

[0014] While the technologies described in Patent Documents 3 and 4 had the advantage of being able to control the culture space in three dimensions, there was a concern that the cultured cells might be damaged when removing them because the multiple bonded components could not be separated.

[0015] The inventors of this invention believed that in order to culture organoids that have the same function as tissues in the body, it was important to develop a dedicated culture vessel that could simulate cell growth in vivo. Therefore, one aspect of the present invention aims to provide a culture vessel that has sufficient oxygen supplied from the bottom of the culture vessel to which cultured substances such as cells adhere, a culture space suitable for three-dimensional control of the growth of the cultured substances, and further, a vessel that allows grown cultured substances to be removed without damage. [Means for solving the problem]

[0016] The inventors diligently studied to solve the above problems. As a result, they found that the above problems can be solved with the culture vessel, method for manufacturing the same, and culture method described below, and thus completed the present invention. One aspect of the present invention is, for example, the following [1] to

[26] .

[0017] [1] A culture vessel having a culture space for culturing a substance to be cultured, wherein the culture vessel comprises a container member (B) having an inverted recess on the lower surface of the bottom and a first through hole at one end of the inverted recess, and an oxygen-permeable film member (A), the film member (A) and the container member (B) being peelably attached to each other, and in the state in which the film member (A) and the container member (B) are attached, a space α is formed by the inverted recess and the film member (A), the first through hole located at one end of the space α receives the substance to be cultured, and the space α is part of the culture space. [2] The culture vessel according to [1], wherein the space α is a flow channel.

[0018] [3] The culture vessel according to [1] or [2], wherein the width of the channel is in the range of 1 μm to 1000 μm and the depth is in the range of 1 μm to 1000 μm. [4] The culture vessel according to [2] or [3], wherein the length of the flow channel is in the range of 1 mm to 100 mm.

[0019] [5] The culture vessel according to any one of [1] to [4], wherein the thickness of the film member (A) is in the range of 1 μm to 1000 μm. [6] The culture vessel according to any one of [1] to [5], wherein the inverted recess is an inverted recess for flow path. [7] A culture container according to any one of [1] to [6], wherein the peel strength in a 90° peel test between the film member (A) and the container member (B), measured in accordance with JIS-K6854-1:1999, is in the range of 0.01 to 3.0 N / cm.

[0020] [8] A culture vessel according to any one of [1] to [7], wherein the total light transmittance of the film member (A), as measured in accordance with JIS-K-7361-1, is 80% or more. [9] The oxygen permeability of the film member (A) at a temperature of 23°C and a humidity of 0% is 4500 to 90000 cm 3 / (m 2 A culture vessel described in any of [1] to [8], within the range of ×24h × atm.

[0021]

[10] The culture container according to any one of [1] to [9], wherein the container member (B) contains resin.

[11] The culture vessel according to

[10] , wherein the resin is a thermoplastic resin.

[12] The culture vessel according to

[11] , wherein the thermoplastic resin has a glass transition temperature of 120°C or less, as measured in accordance with JIS-K7121:1987.

[0022]

[13] The culture vessel according to any one of [1] to

[12] , wherein the film member (A) comprises a 4-methyl-1-pentene polymer (X).

[14] The culture vessel according to any one of [1] to

[13] , wherein the container member (B) comprises a 4-methyl-1-pentene polymer (X).

[15] The culture vessel according to

[13] or

[14] , wherein the 4-methyl-1-pentene polymer (X) is at least one polymer selected from a 4-methyl-1-pentene homopolymer (X1) and a copolymer (X2) of 4-methyl-1-pentene with at least one olefin selected from ethylene, propylene, and α-olefins having 4 to 20 carbon atoms.

[0023]

[16] A culture container according to any one of [1] to

[15] , wherein the water contact angle of at least the culture surface of either the film member (A) or the container member (B) is 50° to 100°.

[17] The culture container according to any one of [1] to

[16] , wherein at least one culture surface of either the film member (A) or the container member (B) is coated with at least one material selected from the group consisting of natural polymer materials, synthetic polymer materials, and inorganic materials.

[0024]

[18] The culture container according to any one of [1] to

[17] , wherein at least one of the culture surfaces of the film member (A) and the container member (B) is a culture surface that has been treated to be hydrophilic.

[19] A culture vessel according to any one of [1] to

[18] , wherein the other end of the inverted recess is provided with a second through hole, and the other end of the space α communicates with the second through hole.

[0025]

[20] The culture container according to any one of [1] to

[19] , wherein the container member (B) has a liquid reservoir for containing a culture medium, and the liquid reservoir, the first through hole, the second through hole, and the space α are in communication with each other.

[21] A culture vessel according to any one of [1] to

[20] , wherein the cultured material is nerve cells or cardiomyocytes.

[22] The culture vessel according to

[21] , wherein the first through-hole is a first chamber portion for receiving the cultured material, the cell bodies of the nerve cells are received in the first chamber portion, and the axonal bundles extending from the cell bodies are received in the space α.

[0026] A method for culturing cells, tissues, or organs, comprising the step (I) of incubating cells, tissues, or organs in a culture vessel described in any of

[23] [1] to

[22] .

[24] The culture method according to

[23] , further comprising: a peeling step (II) for peeling the film member (A) and the container member (B) after step (I); and a removal step (III) for removing the incubated cells, tissue, or organ after step (II).

[0027] A method for manufacturing a culture vessel according to any one of

[25] [1] to

[22] , comprising a bonding step of peelably bonding a container member (B) having an inverted recess on the lower surface of the bottom and a first through hole at one end of the inverted recess, to an oxygen-permeable film member (A).

[26] The method for manufacturing a culture vessel according to

[25] , wherein the bonding step comprises a step of hydrophilizing part or all of the bonding surfaces of the film member (A) and the container member (B), and then heat-pressing the hydrophilized bonding surfaces, or a laser irradiation step of irradiating the bonding surface of the film member (A) and the bonding surface of the container member (B) with a laser while they are in contact. [Effects of the Invention]

[0028] According to one aspect of the present invention, a culture vessel is provided to which sufficient oxygen for growth is supplied from the bottom of the culture vessel to which the cultured material such as cells adheres, which has a culture space suitable for three-dimensional control of the growth of cells, and which allows the cultured cells to be removed without damage by detaching the components after culture.

[0029] According to one aspect of the present invention, it is possible to provide a culture vessel that can culture cells, tissues, or organs in an environment that mimics the environment inside a living body, and further, a method for producing the same, and a method for culturing cells, tissues, or organs that can be removed without damage. [Brief explanation of the drawing]

[0030] [Figure 1] Figure 1 shows a top view, cross-sectional view, and plan view of the lower part of the bottom of a preferred example of container member (B), as well as three-dimensional views of the container member (B) from the top and bottom sides. (1) Plan view of the top, (2) Cross-sectional view of the area indicated by the dashed lines in (1) and (3), (3) Plan view of the lower part of the bottom, (4) Three-dimensional view from the top side, (5) Three-dimensional view from the bottom side [Figure 2] Figure 2 shows an example of a suitable culture container in which a film member (A) and a container member (B) are peelably attached. (6) Details of the cross-section at the location indicated by the dashed line in Figure 1(1) and (3) above, (7) Stereoscopic view from the bottom side, (8) Stereoscopic view from the top side; When culturing a cultured organism using a culture container according to one embodiment of the present invention, it is preferably used in the state of (8). [Figure 3] Figure 3 shows images of axonal bundles of nerve cells (spheroid 1) cultured using a culture vessel according to one embodiment of the present invention, observed with an optical microscope through a film member (A) attached to the bottom surface of the culture vessel. (9) shows culture vessel 1, (10) shows culture vessel 3, and (11) shows culture vessel 4. [Modes for carrying out the invention]

[0031] <Culture container> In one embodiment of the present invention, "culture vessel" means all containers used for culturing cells, tissues, or organs in a culture medium or culture solution. To culture in a culture medium or culture solution means that at least a portion of the cells, etc., are cultured in contact with the culture medium or culture solution, and the entire cells, etc., to be cultured do not necessarily have to be immersed in the culture medium or culture solution. Being able to remove cultured material without damage means that, when observed under a light microscope, the shape of the cultured cells, etc., remains virtually unchanged before and after removal from the culture vessel.

[0032] A culture vessel according to one aspect of the present invention is a culture vessel having a culture space for culturing a substance to be cultured, wherein the culture vessel comprises a container member (B) having an inverted recess on the lower surface of its bottom and a first through hole at one end of the inverted recess, and an oxygen-permeable film member (A), wherein the film member (A) and the container member (B) are detachably attached to each other, and in the state in which the film member (A) and the container member (B) are attached, a space α is formed by the inverted recess and the film member (A), the first through hole located at one end of the space α receives the substance to be cultured, and the space α is part of the culture space. A culture vessel according to one aspect of the present invention may have members other than the film member (A) and the container member (B), and may also be equipped with stirring members such as stirring blades and baffles for stirring.

[0033] [Film component (A)] The film member (A) is oxygen permeable. In one embodiment of the present invention, the culture vessel is used with the film member (A) and the container member (B), which will be described later, attached to each other. One embodiment of the present invention provides a culture vessel having a film member (A) with appropriate oxygen permeability when culturing cells with different oxygen requirements, by controlling the oxygen permeability of the film member (A).

[0034] Conventional techniques have made it difficult to adequately supply oxygen to cells in the culture medium for cells with high oxygen requirements. However, in one embodiment of the present invention, by selecting a highly oxygen-permeable material for the film member (A), a suitable culture vessel can be provided even for cells with high oxygen requirements.

[0035] (Oxygen permeability) In one aspect of the present invention, having oxygen permeability means that the oxygen permeability at a temperature of 23°C and a humidity of 0% is greater than 0.01 cm 3 / (m 2 ×24 h×atm). The oxygen permeability of the film member (A) at a temperature of 23°C and a humidity of 0% is preferably in the range of 0.1 to 1,000,000 cm 3 / (m 2 ×24 h×atm), more preferably in the range of 1 to 1,000,000 cm 3 / (m 2 ×24 h×atm), still more preferably in the range of 10 to 1,000,000 cm 3 / (m 2 ×24 h×atm), even more preferably in the range of 100 to 1,000,000 cm 3 / (m 2 ×24 h×atm), still even more preferably in the range of 1,000 to 90,000 cm 3 / (m 2 ×24 h×atm), and most preferably in the range of 4,500 to 90,000 cm 3 / (m 2 ×24 h×atm). When the oxygen permeability is 4,500 to 90,000 cm 3 / (m 2 ×24 h×atm), the oxygen concentration in the culture medium is appropriate, and cells and the like can grow and differentiate sufficiently and grow. Also, it is preferable because it is possible to avoid a decrease in cell function due to oxygen stress.

[0036] When culturing cells with high oxygen requirements, specifically, hepatocytes, renal cells, cardiomyocytes, and neurons, the oxygen permeability of the film member (A) is preferably in the range of 45,000 to 90,000 cm 3 / (m 2 ×24 h×atm).

[0037] The oxygen permeability can be measured in accordance with JIS-K-7126-1 using a differential pressure method gas permeability measuring device (manufactured by Toyo Seiki Seisakusho).

[0038] (Total light transmittance) In a culture vessel according to one aspect of the present invention, the film member (A) preferably has a total light transmittance of 80% or more, more preferably 85.0% or more, and even more preferably 90.0% or more, as measured in accordance with JIS-K-7361-1. There is no particular upper limit to the total light transmittance, but it is usually 99.9%. If the total light transmittance is 80% or higher, it can be said that the material has excellent transparency.

[0039] A total light transmittance of 80% or more is preferable because it provides high transparency to the film component (A), making it easier to observe the cultured material in the culture vessel and to observe cells and other elements both with the naked eye and under a microscope.

[0040] The film member (A) preferably has a thickness in the range of 1 μm to 1000 μm, more preferably in the range of 10 μm to 500 μm, and even more preferably in the range of 20 μm to 200 μm. When the thickness of the film member (A) is within the above range, it is possible to provide sufficient strength so that the film member (A) is not damaged during peeling, and to supply oxygen suitable for cultivation to the cultured material. In other words, it is preferable because it is possible to achieve both strength and oxygen permeability of the film member (A).

[0041] The film member (A) only needs to be large enough to adhere to the container member (B) described later, and its size is not particularly limited, however, from the viewpoint of ease of peeling, it is preferable that the film member (A) is larger than the area of ​​the bottom of the container member (B).

[0042] (water contact angle) The film member (A) has a water contact angle of at least 30° or more, more preferably 35° or more, even more preferably 40° or more, particularly preferably 50° or more, preferably 120° or less, more preferably 110° or less, and even more preferably 100° or less on the culture surface. The film member (A) is particularly preferably such that the water contact angle of the culture surface is at least 50° to 100°.

[0043] In one embodiment of the present invention, the culture surface refers to the portion of the culture vessel that is in contact with the culture medium and / or cells when culturing cells or the like as the subject to be cultured, or the portion that is intended to be in contact with the culture medium and / or cells. In other words, it refers to the portion of the inside of the culture vessel that is in contact with the culture medium and / or cells.

[0044] Adjusting the water contact angle of the film member (A) to the above range is preferable because it makes it easier for cells, for example, to adhere to the film member (A), and allows for uniform proliferation of cells, etc., on the film member (A). Furthermore, when a natural polymer material, synthetic polymer material, or inorganic material is uniformly coated onto the film member (A), it becomes easier to adhere the cultured material. In addition, even after coating, the natural polymer material, synthetic polymer material, or inorganic material does not peel off during washing with physiological saline and in the cell culture environment, maintaining a stable initial state and being usable for cell culture, which is preferable.

[0045] The method for measuring the water contact angle is not particularly limited, and known methods can be used, but the static droplet method is preferred. The water contact angle can be measured, for example, in accordance with JIS-R3257 (Test method for wettability of substrate glass surfaces) as described below.

[0046] (Test method for wettability of substrate glass surface) In accordance with JIS-R3257 (Test method for wettability of substrate glass surfaces), a water droplet of 4 μL or less, whose shape can be considered spherical, is dropped onto the surface of the sample to be measured under constant temperature and humidity conditions of 25 ± 5°C and 50 ± 10%, and the angle of the contact interface between the sample and the water droplet is measured using the static drop method, from immediately after the water droplet contacts the sample surface to within 1 minute.

[0047] The film member (A) is not particularly limited as long as it is oxygen permeable, but the material of the film member (A) is preferably one that has excellent moldability so that it can be made into various shapes depending on the application. Examples of materials for the film member (A) include resins, carbon materials such as carbon paper, and porous inorganic materials such as ceramics. Among these, it is preferable that the film member (A) contains a resin. Furthermore, it is even more preferable that the film member (A) is a thermoplastic resin.

[0048] The thermoplastic resin is not particularly limited, but examples include: polyolefin resins; polymethacrylic resins such as polymethyl methacrylate resin; polyacrylic resins such as polymethyl acrylate resin; polystyrene resins; polyvinyl acetal resins; polyvinyl butyral resins; polyvinyl formal resins; polymethylpentene resins; polycarbonate resins; polyether ether ketone resins; polyether ketone resins; polyester resins; polyamide resins such as nylon-6, nylon-66, and polymetaxylene adipamide; polyamide-imide resins; polyimide resins; polyetherimide resins; styrene elastomers; Examples include polyolefin elastomers; polyurethane elastomers; polyester elastomers; polyamide elastomers; norbornene resins; polytetrafluoroethylene resins; ethylene tetrafluoroethylene copolymers; polyvinylidene fluoride resins; polyvinyl fluoride resins; thermoplastic polyimide resins; polyvinylidene chloride resins; polyvinyl chloride resins; polyvinyl acetate resins; polysulfone resins; polyphenylene resins such as polyphenylene oxide resins and polyphenylene sulfide resins; polysulfone resins; polylactic acid resins; polyethersulfone resins; polyacrylonitrile resins, styrene-acrylonitrile copolymer resins, and the like.

[0049] Among these, it is preferable that the film member (A) is at least one selected from polyolefin resins and fluororesins, with polyolefin resins being more preferable.

[0050] Examples of polyolefin resins include homopolymers or copolymers of α-olefins such as ethylene, propylene, 1-butene, 1-hexene, 4-methyl-1-pentene, and 1-octene; high-pressure low-density polyethylene; linear low-density polyethylene (LLDPE); high-density polyethylene; polypropylene; copolymers of propylene and α-olefins having 2 to 10 carbon atoms; ethylene / vinyl acetate copolymer (EVA); ionomer resins; and fluorine-containing cyclic olefin polymers.

[0051] Examples of fluororesins include polytetrafluoroethylene (PTFE), polytrifluorochloroethylene, polyvinyl fluoride, polyvinylidene fluoride, dichlorodifluoroethylene, polychlorotrifluoroethylene, fluorinated ethylene propylene copolymer, perfluoroalkyl vinyl ether polymer, perfluoroalkyl vinyl ester polymer, and ethylene tetrafluoroethylene copolymer.

[0052] Among these, at least one selected from 4-methyl-1-pentene polymer and polystyrene is more preferred, as it offers an excellent balance of moldability, transparency, shape stability, lightness, and low drug sorption, as well as excellent release properties between the film member (A) and the container member (B). The 4-methyl-1-pentene polymer (X), described later, is even more preferred. In other words, in one embodiment of the present invention, the culture vessel comprises a film member (A), which preferably contains a 4-methyl-1-pentene polymer (X). The material of the film component (A) may consist of one type alone or two or more types.

[0053] [4-methyl-1-pentene polymer] In one embodiment of the present invention, 4-methyl-1-pentene homopolymers and copolymers of 4-methyl-1-pentene with other monomers are collectively referred to as "4-methyl-1-pentene polymer (X)". In a culture vessel according to one aspect of the present invention, the film member (A) and the container member (B) preferably contain a 4-methyl-1-pentene polymer, and more preferably contain a 4-methyl-1-pentene polymer (X). In one embodiment of the present invention, the culture vessel, the film member (A) and the container member (B) may be formed solely from a 4-methyl-1-pentene polymer (X), or they may be formed from a composition containing a 4-methyl-1-pentene polymer (X).

[0054] An example of a 4-methyl-1-pentene polymer (X) is a copolymer of 4-methyl-1-pentene and another monomer, which may be a random copolymer, an alternating copolymer, a block copolymer, or a graft copolymer. As the copolymer of 4-methyl-1-pentene and another monomer, a copolymer of 4-methyl-1-pentene and at least one olefin selected from ethylene and α-olefins having 3 to 20 carbon atoms (excluding 4-methyl-1-pentene) is preferred because it has high strength, i.e., it is resistant to tearing and cracking and has little bending.

[0055] The 4-methyl-1-pentene polymer (X) is preferably at least one polymer selected from a 4-methyl-1-pentene homopolymer and a copolymer of 4-methyl-1-pentene and ethylene and at least one olefin selected from α-olefins having 3 to 20 carbon atoms (excluding 4-methyl-1-pentene), and more preferably a copolymer of 4-methyl-1-pentene and ethylene and at least one olefin selected from α-olefins having 3 to 20 carbon atoms (excluding 4-methyl-1-pentene).

[0056] Examples of the olefins mentioned above include ethylene, propylene, 1-butene, 1-hexene, 1-heptene, 1-octene, 1-decene, 1-tetradecene, 1-hexadecene, 1-heptadecene, 1-octadecene, and 1-eicosene. The olefin can be appropriately selected according to the physical properties required for the film member (A) and the container member (B). For example, as the olefin, α-olefins having 8 to 18 carbon atoms are preferred from the viewpoint of moderate oxygen permeability and excellent rigidity, and at least one selected from 1-octene, 1-decene, 1-dodecene, 1-tetradecene, 1-hexadecene, 1-heptadecene, and 1-octadecene is more preferred. When the carbon number of the olefin is within the above range, the moldability of the polymer is improved, and as a result, defects in appearance due to cracks or cracks at the edges during release from the roll and mold during molding tend to occur less frequently. Therefore, the rate of defective products of the film member (A) and the container member (B) is reduced.

[0057] The aforementioned olefin may consist of only one type, or it may be a combination of two or more types. From the viewpoint of material strength, the number of carbon atoms is preferably 2 or more, and more preferably 10 or more. When combining two or more different α-olefins, it is particularly preferable to combine at least one selected from 1-tetradecene and 1-hexadecene with at least one selected from 1-heptadecene and 1-octadecene.

[0058] The content of the constituent 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 100 mol%, and even more preferably 85 to 99 mol%. Furthermore, if the 4-methyl-1-pentene polymer (X) is a copolymer of 4-methyl-1-pentene and ethylene and at least one olefin selected from α-olefins having 3 to 20 carbon atoms (excluding 4-methyl-1-pentene), the content of constituent units derived from ethylene and at least one olefin selected from α-olefins having 3 to 20 carbon atoms (excluding 4-methyl-1-pentene) in the copolymer is preferably 0 to 40 mol%, more preferably 0 to 20 mol%, and even more preferably 1 to 15 mol%. Note that the content of these constituent units is defined as 100 mol% of the total repeatable constituent units in the 4-methyl-1-pentene polymer (X). A constituent unit content within the above range is preferable because it provides excellent processability, a homogeneous culture surface, and a good balance between the toughness and strength of the film, resulting in less bending.

[0059] The 4-methyl-1-pentene polymer (X) may have structural units other than those derived from 4-methyl-1-pentene and those derived from ethylene and α-olefins having 3 to 20 carbon atoms (hereinafter also referred to as "other structural units"), to the extent that the effects of the present invention are not impaired. The content of other structural units is, for example, 0 to 10.0 mol%. If the 4-methyl-1-pentene polymer (X) has other structural units, there may be one or more types of other structural units.

[0060] Examples of monomers used to derive other constituent units include cyclic olefins, aromatic vinyl compounds, conjugated dienes, unconjugated polyenes, functional vinyl compounds, hydroxyl group-containing olefins, and halogenated olefins. For example, compounds described in paragraphs

[0035] to

[0041] of Japanese Patent Application Publication No. 2013-169685 can be used as cyclic olefins, aromatic vinyl compounds, conjugated dienes, unconjugated polyenes, functional vinyl compounds, hydroxyl group-containing olefins, and halogenated olefins.

[0061] The 4-methyl-1-pentene polymer (X) may be used alone or in combination of two or more types.

[0062] Commercially available 4-methyl-1-pentene polymers (X) can also be used. Specifically, examples include TPX MX001, MX002, MX004, MX0020, MX021, MX321, RT18, RT31, or DX845 (all trademarks) manufactured by Mitsui Chemicals, Inc. In addition, 4-methyl-1-pentene polymers (X) from other manufacturers that meet the above requirements can also be preferably used. These commercial products may be used individually or in combination of two or more.

[0063] When a film member (A) and a container member (B) according to one embodiment of the present invention are formed from a composition containing a 4-methyl-1-pentene polymer (X), other components may be included. Examples of other components include heat-resistant stabilizers, light-resistant stabilizers, processing aids, plasticizers, antioxidants, lubricants, defoamers, antiblocking agents, colorants, modifiers, antibacterial agents, antifungal agents, and antifogging agents.

[0064] The 4-methyl-1-pentene polymer (X) typically has a melting point of 200°C to 240°C and exhibits high heat resistance. Furthermore, it does not undergo hydrolysis and has excellent water resistance, boiling water resistance, and steam resistance. Therefore, the film component (A) and container component (B) containing the 4-methyl-1-pentene polymer (X) can be subjected to high-pressure steam sterilization. The 4-methyl-1-pentene polymer (X) has high visible light transmittance (usually 90% or more) and does not emit autofluorescence, so the film material (A) containing the 4-methyl-1-pentene polymer (X) facilitates observation of cultured organisms. Furthermore, it exhibits excellent chemical resistance to most drugs and is resistant to drug sorption, so as a culture medium, it does not interfere with the effects of drugs used to maintain cells, for example, and is also suitable for drug discovery screening and diagnostic applications.

[0065] Because the 4-methyl-1-pentene polymer (X) has high strength, culture vessels having a film member (A) and a container member (B) containing the 4-methyl-1-pentene polymer (X) are less prone to cracking and less prone to bending. The 4-methyl-1-pentene polymer (X) is heat-sealable and readily heat-bonds not only to itself but also to other materials. Furthermore, because the 4-methyl-1-pentene polymer (X) is thermoformable, it can be easily molded into culture vessels of any shape, and can be easily molded using methods such as imprinting or inserting.

[0066] The weight-average molecular weight (Mw) of the 4-methyl-1-pentene polymer (X) is preferably 10,000 to 2,000,000, more preferably 20,000 to 1,000,000, and even more preferably 30,000 to 500,000, as determined by the number-average molecular weight (GPC) measurement method described below. Furthermore, 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, and even more preferably 1.1 to 20, as determined by the GPC method described below.

[0067] (GPC measurement method) GPC measurement is performed at a temperature of 140°C using orthodichlorobenzene as the solvent, and analytical values ​​(weight-average molecular weight (Mw), number-average molecular weight (Mn), and Mw / Mn) can be obtained as polyethylene equivalent values. The sample concentration during GPC measurement can be, for example, 1.0 to 5.0 mg / ml.

[0068] Measurements can be performed under the following conditions. Furthermore, the molecular weight can be determined by creating a calibration curve using commercially available monodisperse standard polystyrene and calculating it based on the conversion method described below. Equipment: Gel permeation chromatograph Alliance GPC2000 (Waters Corporation) Solvent: o-dichlorobenzene Columns: TSKgel columns (manufactured by Tosoh Corporation) x 4 Flow rate: 1.0ml / min Sample: 0.15 mg / mL of dichlorobenzene solution Temperature: 140℃ Molecular weight conversion: PS conversion / General calibration method

[0069] Furthermore, the coefficients of the Mark-Houwink viscosity equation shown below can be used in calculations using the general calibration method. Coefficients for polystyrene (PS): KPS = 1.38 × 10⁻⁴, aPS = 0.70 Coefficients for polyethylene (PE): KPE = 5.06 × 10⁻⁴, aPE = 0.70

[0070] The solvent used in the above GPC measurement method is not particularly limited as long as it dissolves the 4-methyl-1-pentene polymer (X), but orthodichlorobenzene is preferred.

[0071] By keeping the weight-average molecular weight (Mw) below the above upper limit, when manufacturing the film member (A) by melt molding in the above-described molding method for the 4-methyl-1-pentene polymer (X), it is preferable because it is easier to suppress the occurrence of defects such as gel and to form a uniform film on the surface. Furthermore, when manufacturing the film member (A) by the solution casting method, it is preferable because it improves solubility in the solvent, makes it easier to suppress defects such as gel in the film member (A), and facilitates the formation of a uniform film on the surface.

[0072] By setting the weight-average molecular weight (Mw) to be above the lower limit, when manufacturing the container member (B) by injection molding in the above-described molding method for the 4-methyl-1-pentene polymer (X), the strength of the container member (B) tends to be sufficient. Furthermore, by keeping the molecular weight distribution within the above range, it is easier to suppress stickiness on the surface of the manufactured container member (B), and the toughness of the container member (B) tends to be sufficient, which is preferable because it makes it easier to suppress the occurrence of cracks during bending and cutting during molding.

[0073] The weight-average molecular weight (Mw) and molecular weight distribution (Mw / Mn) of the 4-methyl-1-pentene polymer (X) are such that, if two or more types of 4-methyl-1-pentene polymers (X) are used, the Mw and Mw / Mn of each must fall within the above range.

[0074] In one embodiment of the present invention, the inclusion of the 4-methyl-1-pentene polymer (X) in the film member (A) and the container member (B) is considered to have virtually no adverse effect on the culture. Since the 4-methyl-1-pentene polymer (X) has the above-mentioned excellent properties, when the film member (A) contains the 4-methyl-1-pentene polymer (X), it exhibits good shape stability, transparency, sheet-like processability, and oxygen permeability. Furthermore, when the container member (B) contains the 4-methyl-1-pentene polymer (X), it exhibits good shape stability, transparency, and moldability. Furthermore, since a culture vessel according to one aspect of the present invention can be sterilized, it is an excellent culture vessel for culturing cells or the like as a cultured material.

[0075] In one embodiment of the present invention, the term "polymer" is used to encompass both homopolymers and copolymers. Similarly, in one embodiment of the present invention, the term "polymerization" is used to encompass both homopolymerization and copolymerization.

[0076] The thermoplastic resin used as the material for the film member (A) preferably has a glass transition temperature of 200°C or lower, more preferably 150°C or lower, and even more preferably 120°C or lower, as measured in accordance with JIS-K7121:1987. A glass transition temperature of 200°C or lower is preferable because it makes it easier to mold into a fine structure. The lower limit of the glass transition temperature is not particularly limited, but it can be, for example, 0°C or higher.

[0077] The surface shape of the film member (A) is not particularly limited. The film member (A) can have an uneven surface pattern so that when it is attached to the container member (B), the uneven portion can become part of the space α. If the surface to which the film member (A) and the container member (B) are adhered is not smooth, it may cause a decrease in peel strength. Therefore, it is preferable that the film member (A) does not have an uneven surface pattern.

[0078] [Container component (B)] The container member (B) has an inverted recess on the lower surface of its bottom, and a first through hole is provided at one end of the inverted recess. The container member (B) preferably has a second through hole at the other end of the inverted recess. In other words, the bottom surface of the container member (B) is provided with a first through-hole and a second through-hole, so that the culture medium necessary for cultivation can be circulated, for example, from the first through-hole to the second through-hole, thereby maintaining a constant culture medium composition. Furthermore, it is preferable because it can also be used for cell culture in which two different types of cultured organisms, such as cells, are seeded in the first and second through-holes respectively and joined together.

[0079] The container member (B) preferably has an open top. Having an open top allows for easy supply of culture medium to the liquid reservoir and removal of culture medium from the liquid reservoir during culture medium replacement without affecting the cultured cells or other cultured substances.

[0080] The upper bottom surface of container member (B) is preferably used as a liquid reservoir for containing the culture medium. This structure allows for a compact culture container without the need for a separate liquid reservoir, thus avoiding a complex structure. Preferably, the container member (B) has a liquid reservoir for containing the culture medium, and the liquid reservoir, the first through-hole, the second through-hole, and space α are in communication with each other. With such a structure, for example, the culture medium can be circulated from the first through-hole through space α to the second through-hole, and by supplying space α with a culture medium of a constant composition, the culture conditions can be kept constant.

[0081] The size of the container member (B) is not particularly limited, but from the viewpoint of improving operability in removing the cultured material, for example, the length and width are preferably in the range of 0.1 to 200 cm, more preferably in the range of 0.3 to 100 cm, and even more preferably in the range of 0.5 to 30 cm.

[0082] Since oxygen supply to space α is achieved by controlling the oxygen permeability of the film component (A), it is desirable that the volume ratio of the container component (B) to the volume of space α ([volume of container component (B)] / [volume of space α]) be above a certain size so that the oxygen supply from the container component (B) to space α does not have a significant impact. On the other hand, from the viewpoint of volume efficiency as a culture vessel, it is desirable that the volume ratio of the container component (B) to the volume of space α not become too large. Taking these factors into consideration, the volume ratio of the container member (B) to space α ([volume of container member (B)] / [volume of space α]) is preferably 1 to 100,000,000, more preferably 1 to 1,000,000, and even more preferably 10 to 10,000.

[0083] (Inverted recess) The shape of the inverted recess on the bottom surface of the container member (B) is not particularly limited. Examples of inverted recess shapes include semicircular, semielliptical, rectangular, square, trapezoidal, inverted V-shaped, and inverted U-shaped cross-sections. Among these, when the cultured material is nerve cells, the shape of the inverted recess is preferably semicircular, inverted U-shaped, trapezoidal, or square in cross-section, as this allows the axons to easily extend along the inverted recess and makes it easy to remove the cultured material without damage. It is preferable that the inverted recess on the bottom surface of the container member (B) is an inverted recess for flow path. In other words, when the film member (A) and the container member (B) are attached to each other, it is preferable that the portion formed by the inverted recess on the bottom surface of the container member (B) and the film member (A) is used as a flow path. Furthermore, the shape of the inverted recess for the flow path is more preferably semicircular, inverted U-shaped, trapezoidal, or square in cross-section.

[0084] The size of the inverted recess on the bottom surface of the container member (B) is not particularly limited, but the maximum vertical depth of the inverted recess is preferably in the range of 1 to 1000 μm, more preferably 10 to 1000 μm, more preferably 20 to 200 μm, more preferably 30 to 180 μm, and even more preferably 50 to 100 μm. The maximum horizontal width is preferably in the range of 10 to 1000 μm, more preferably 20 to 200 μm, more preferably 30 to 180 μm, and even more preferably 50 to 100 μm.

[0085] The length of the inverted recess on the bottom surface of the container member (B) in the direction of extension is not particularly limited, but when the cultured material is nerve cells, it is preferable that it be long enough to accommodate an axon bundle of the desired length. The length of the inverted recess on the bottom surface in the direction of extension is preferably 1 to 50 mm, more preferably 5 to 30 mm, more preferably 5 to 20 mm, and even more preferably 5 to 15 mm.

[0086] The inverted recess on the bottom surface of the container member (B) is preferably a groove with a semicircular or square cross-section, with a maximum vertical depth of 50-100 μm, a maximum horizontal width of 50-100 μm, and a length in the extension direction of 5-15 mm. This shape is particularly preferable because, when the cultured material is nerve cells, the axons can easily extend along the channel, and the cultured material can be easily removed without damage.

[0087] (first through hole) The container member (B) has an inverted recess on the lower surface of its bottom, and a first through hole is provided at one end of the inverted recess. The first through-hole becomes the first chamber when the film member (A) and the container member (B) are attached to each other. The first chamber has a role, for example, in receiving the cultured material.

[0088] The shape of the first through-hole is not particularly limited, but is preferably cylindrical. The size of the first through-hole is not particularly limited as long as it can accommodate the cultured material, but is preferably 0.1 to 10 mm in diameter, more preferably 0.5 to 5 mm in diameter, and even more preferably 0.8 to 2 mm in diameter.

[0089] (Second through hole) The container member (B) preferably has a second through-hole. The second through-hole becomes a second chamber when the film member (A) and the container member (B) are attached to each other. The second chamber serves, for example, as a vent and an outlet for circulating the culture medium. The shape of the second through-hole is not particularly limited, but is preferably cylindrical. The size of the second through-hole is not particularly limited as long as it can accommodate the cultured material, but is preferably 0.1 to 10 mm in diameter, more preferably 0.1 to 5 mm in diameter, and even more preferably 0.3 to 2 mm in diameter. The second through-hole does not need to be the same size as the first through-hole, as long as it allows for efficient aeration and drainage of the culture medium. In particular, when used in cultures where it is necessary to secure sufficient space for the cultured material, it is preferable that the first through-hole be larger than the second through-hole.

[0090] A module is defined as a part of a reverse concave section that has a first through-hole at one end. Preferably, the module has a second through-hole. There are no particular restrictions on the number of modules, but the container member (B) preferably comprises 1 to 100 modules, more preferably 1 to 50 modules, and even more preferably 1 to 20 modules. The container member (B) only needs to have a module, and there are no particular restrictions on the size and shape of the container member (B).

[0091] The bottom surface of the container member (B) is provided with a first through hole with a diameter of 0.8 to 2 mm, and further with a second through hole with a diameter of 0.3 to 2 mm, and it is more preferable that there be 1 to 40 of each of the first and second through holes.

[0092] (Liquid reservoir) The liquid reservoir can contain the culture medium when the film member (A) and the container member (B) are attached to each other. The volume of the liquid reservoir is preferably 0.1 to 10,000 mL, more preferably 0.5 to 1,000 mL, and even more preferably 1 to 100 mL. The volume of the culture medium to be contained is not particularly limited, but is preferably 0.1 to 10,000 mL, more preferably 0.5 to 1,000 mL, and even more preferably 1 to 100 mL.

[0093] The liquid reservoir is preferably used as a section for storing culture medium to supply it to space α. When storing culture medium in the liquid reservoir, the volume ratio of the liquid reservoir to the volume of space α must be above a certain size. On the other hand, when removing the cultured material from space α, it becomes necessary to drain, for example, the culture medium accumulated in the liquid reservoir. Therefore, from the viewpoint of operability, it is desirable to prevent the volume ratio of the liquid reservoir to the volume of space α from becoming too large. Taking these factors into consideration, the volume ratio of the liquid reservoir to space α ([volume of the liquid reservoir] / [volume of space α]) is preferably 1 to 100,000,000, more preferably 1 to 1,000,000, and even more preferably 10 to 500,000.

[0094] In the liquid reservoir, it is preferable that the first through-hole, the second through-hole, and the space α are in communication when the film member (A) and the container member (B) are attached to each other.

[0095] The material for the container component (B) is preferably one that has moldability so that it can be made into various shapes depending on the application. In particular, good moldability that can accommodate fine structures is important. Examples of materials for the container component (B) include resin, carbon materials such as carbon paper, and porous inorganic materials such as ceramics. Among these, it is preferable that the container component (B) contains resin. Furthermore, it is even more preferable that the container member (B) contains a resin, and that the resin is a thermoplastic resin.

[0096] The thermoplastic resin is not particularly limited, but examples include: polyolefin resins; polymethacrylic resins such as polymethyl methacrylate resin; polyacrylic resins such as polymethyl acrylate resin; polystyrene resins; polyvinyl acetal resins; polyvinyl butyral resins; polyvinyl formal resins; polymethylpentene resins; polycarbonate resins; polyether ether ketone resins; polyether ketone resins; polyester resins; polyamide resins such as nylon-6, nylon-66, and polymetaxylene adipamide; polyamide-imide resins; polyimide resins; polyetherimide resins; styrene elastomers; and Examples include polyolefin elastomers; polyurethane elastomers; polyester elastomers; polyamide elastomers; norbornene resins; polytetrafluoroethylene resins; ethylenetetrafluoroethylene copolymers; polyvinylidene fluoride resins; polyvinyl fluoride resins; thermoplastic polyimide resins; polyvinylidene chloride resins; polyvinyl chloride resins; polyvinyl acetate resins; polysulfone resins; polyphenylene resins such as polyphenylene oxide resins and polyphenylene sulfide resins; polysulfone resins; polylactic acid resins; polyethersulfone resins; polyacrylonitrile resins, styrene-acrylonitrile copolymer resins, and the like.

[0097] Among these, the container member (B) is preferably at least one selected from polyolefin resins and fluororesins, with polyolefin resins being more preferred.

[0098] When using polyolefin resins and fluororesins as the material for the container component (B), the same material as that used for the film component (A) can be used.

[0099] As for the material of the container member (B), at least one selected from 4-methyl-1-pentene polymer and polystyrene is more preferred, and the aforementioned 4-methyl-1-pentene polymer (X) is even more preferred, due to its excellent balance of moldability, transparency, shape stability, lightness, and low drug sorption, as well as its excellent release properties between the film member (A) and the container member (B). In other words, in one embodiment of the present invention, the culture vessel comprises a container member (B), which preferably contains a 4-methyl-1-pentene polymer (X). The material of container component (B) may consist of one type alone or two or more types.

[0100] The material for the container member (B) is preferably a thermoplastic resin with a glass transition temperature of 200°C or lower, more preferably 150°C or lower, and even more preferably 120°C or lower, as measured in accordance with JIS-K7121:1987. A glass transition temperature of 200°C or lower is preferable because it makes it easier to mold into a fine structure. The lower limit of the glass transition temperature is not particularly limited, but it can be, for example, 0°C or higher.

[0101] (water contact angle) The container member (B) preferably has a water contact angle of at least 1° to 130° on the culture surface, more preferably 1° to 120°, and even more preferably 1° to 110°. The water contact angle can be measured, for example, in accordance with the Japanese Industrial Standard JIS-R3257 (Test Method for Wettability of Substrate Glass Surfaces).

[0102] [Method for manufacturing film component (A)] The method for manufacturing the film member (A) is not particularly limited, but if the film member (A) contains, for example, a resin, it can be manufactured by the following method. Methods for forming the film member (A) include, for example, injection molding, extrusion molding, press molding, and blow molding. Among these, the inflation method and T-die extrusion method are preferred for forming the film member (A). The film member (A) can be manufactured using general resin molding methods.

[0103] When the film member (A) contains 4-methyl-1-pentene polymer (X), the amount of 4-methyl-1-pentene polymer (X) is preferably 90% or more and less than 100% by mass, more preferably 95% or more and less than 100% by mass, and even more preferably 99% or more and less than 100% by mass, in 100% by mass of the film member (A). This is because including a large amount of components other than 4-methyl-1-pentene polymer (X) may lead not only to a decrease in oxygen permeability but also to a decrease in transparency or strength. Other components besides 4-methyl-1-pentene polymer (X) include additives such as heat stabilizers, light stabilizers, processing aids, plasticizers, antioxidants, lubricants, defoamers, antiblocking agents, colorants, modifiers, antibacterial agents, antifungal agents, and antifogging agents.

[0104] [Manufacturing method for container component (B)] The method for manufacturing the container member (B) is not particularly limited, but if the container member (B) contains, for example, resin, it can be manufactured by the following method. Methods for forming the container member (B) include, for example, injection molding, extrusion molding, press molding, and blow molding. Among these, injection molding is preferred for forming the container member (B). The container member (B) can be manufactured using general resin molding methods.

[0105] When the container member (B) contains 4-methyl-1-pentene polymer (X), the amount of 4-methyl-1-pentene polymer (X) in 100% by mass of the container member (B) 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. This is because including a large amount of components other than 4-methyl-1-pentene polymer (X) may lead not only to a decrease in oxygen permeability but also to a decrease in transparency or strength. Other components besides 4-methyl-1-pentene polymer (X) include additives such as heat stabilizers, light stabilizers, processing aids, plasticizers, antioxidants, lubricants, defoamers, antiblocking agents, colorants, modifiers, antibacterial agents, antifungal agents, and antifogging agents.

[0106] [Peeling] The film member (A) and the container member (B) are attached to each other in a removable manner. "Removable" means that the attached film component (A) and container component (B) can be separated by hand without significantly damaging the shape of either component. The goal is simply to be able to separate the film component (A) and container component (B) without damaging the cultured material. When separating the film member (A) from the container member (B), for example, the film member (A) can be held down with one hand with its bottom surface facing downwards, and the container member (B) can be grasped with one hand and then lifted up to separate them.

[0107] (Peel strength in peel test) To determine whether the cultured material can be removed without damage after the attached film member (A) and container member (B) are separated, the peel strength measured in a peel test in accordance with JIS-K6854-1:1999 can be used as an indicator. In one embodiment of the present invention, the culture vessel preferably has a peel strength in a 90° peel test between the film member (A) and the container member (B), measured in accordance with JIS-K6854-1:1999, in the range of 0.01 to 3.0 N / cm, more preferably 0.05 to 1.0 N / cm, even more preferably 0.05 to 0.5 N / cm, and still more preferably in the range of 0.1 to 0.5 N / cm. A peel strength of 0.01 N / cm or higher is preferable because it makes it difficult for the film member (A) to peel off the container member (B) by its own weight. On the other hand, a peel strength of 3.0 N / cm or lower is preferable because it makes it difficult for the cultured organism to be damaged by the impact during peeling. For example, it is preferable because it makes it difficult for the cultured organism, such as nerve cells, to be damaged by vibrations caused by the peeling sound or by vibrations in the hand holding the culture vessel.

[0108] [Adhesion] The film member (A) and the container member (B) are attached to each other. In one embodiment of the present invention, it is preferable that when the liquid reservoir is filled with an aqueous solution such as ethanol and culture medium, the film member (A) and the container member (B) are attached in such a way that no liquid leaks from the portion where they are attached. In one embodiment of the present invention, "adhesion" means a state in which two parts are bonded together without the use of an adhesive. The bonding can be carried out by pressurizing or heating, for example, by compression bonding, laser irradiation, or heat pressing. Among these methods, heat pressing is preferred because it allows for peeling without damaging the components and achieves a degree of adhesion that prevents leakage over a long period of time. Bonding can be carried out by pressurizing or heating at least one of the film component (A) and the container component (B). The inventors have found that by using a 4-methyl-1-pentene polymer (X), which can achieve highly adhesive bonding, as the material for the film member (A) and the container member (B), and by setting the peel strength of the 90° peel test between the film member (A) and the container member (B), measured in accordance with JIS-K6854-1:1999, to a range of 0.01 to 3.0 N / cm, a more preferable culture container can be provided, allowing the film member (A) and the container member (B) to be peeled off without applying force that would damage them.

[0109] (Surface treatment) In one embodiment of the present invention, it is preferable that at least the culture surface of either the film member (A) or the container member (B) be surface-treated to facilitate adhesion. The surface treatment is preferably performed before the film member (A) and the container member (B) are attached together. Examples of surface treatments include hydrophilization treatment. The method used for hydrophilization treatment is not particularly limited, but examples include corona treatment, plasma treatment, ozone treatment, ultraviolet treatment, chemical vapor deposition, etching, or addition of specific functional groups such as hydroxyl groups, amino groups, sulfone groups, thiol groups, and carboxyl groups, treatment with specific functional groups such as silane coupling, and surface roughening with oxidizing agents. In particular, surface hydrophilization treatment such as ultraviolet treatment, corona treatment, plasma treatment, or ozone treatment is preferred in order to increase the wettability of the culture material surface and enable efficient cell culture. These surface treatments may be performed individually or in combination of two or more types.

[0110] Furthermore, when surface treatment is performed, it is preferable to perform it at least on the culture surface. When plasma treatment is performed, nitrogen, hydrogen, helium, oxygen, argon, etc., are used as the accompanying gas, and preferably at least one gas selected from nitrogen, helium, and argon is chosen. Among these, it is preferable that at least the culture surface of either the film member (A) or the container member (B) is a plasma-treated culture surface, as this allows the surface to be easily bonded and sufficiently hydrophilic.

[0111] Examples of plasma treatments include vacuum plasma treatment, atmospheric pressure plasma treatment, and high-pressure plasma treatment. Among these, in one embodiment of the present invention, it is preferable that at least the culture surface of either the film member (A) or the container member (B) is treated with at least one of the vacuum plasma treatment and atmospheric pressure plasma treatment. Vacuum plasma treatment can be performed, for example, using a commercially available vacuum plasma surface treatment device (manufactured by Diener Electronic). Atmospheric pressure plasma treatment can be performed, for example, using a commercially available atmospheric pressure plasma surface treatment device (manufactured by Sekisui Chemical Co., Ltd.). When performing atmospheric pressure plasma treatment, gases such as nitrogen, hydrogen, helium, oxygen, and argon are used as the accompanying gas, and preferably, at least one gas selected from nitrogen, helium, and argon is chosen.

[0112] Vacuum plasma treatment is preferred for the surface treatment of the film member (A) and the container member (B), particularly from the viewpoint of efficiently modifying the surface of the members.

[0113] In one embodiment of the present invention, it is preferable that at least one of the culture surfaces of the film member (A) and the container member (B) is a hydrophilic culture surface. By hydrophilizing at least the culture surface of either the film component (A) or the container component (B), the wettability of the culture surface is increased. Furthermore, it becomes easier to coat the culture surface with natural polymer materials, synthetic polymer materials, or inorganic materials. The film member (A) is preferable because, at least the culture surface is treated to make it hydrophilic, which improves the adhesion between the film member (A) and the cultured material, allowing the cultured material to grow uniformly on the surface of the film member (A).

[0114] In one embodiment of the present invention, it is preferable that at least one culture surface of either the film member (A) or the container member (B) is coated with at least one material selected from the group consisting of natural polymer materials, synthetic polymer materials, and inorganic materials. One aspect of the present invention is preferable because, after the above-described coating treatment, the culture vessel does not deform, discolor, or peel even after washing with physiological saline after the coating treatment and under the culture environment of the cultured material, and can be used as a culture vessel while maintaining a stable initial state.

[0115] In one embodiment of the present invention, the culture vessel is preferably coated with an extracellular matrix protein. By coating with an extracellular matrix protein, the extracellular matrix protein functions as an adhesion molecule for cells and other organisms, making it easier to provide a scaffold for cell proliferation. This coating treatment can be carried out using commercially available products and is not particularly limited, but for example, a mouse sarcoma-derived protein (Thermo Fisher Scientific, trade name: Geltrex) or human recombinant laminin (Oriental Yeast Co., Ltd., trade name: rLAMININ-5) can be used.

[0116] [Culture space] A culture vessel according to one aspect of the present invention has a culture space. In one aspect of the present invention, a culture vessel is provided in which a film member (A) and a container member (B) having an inverted recess on the lower surface of the bottom are attached to each other, and a space α is formed by the inverted recess and the film member (A), the first through hole located at one end of the space α receives the cultured material, the space α is part of the culture space, a second through hole is provided at the other end of the inverted recess, and the other end of the space α communicates with the second through hole.

[0117] In one embodiment of the present invention, the culture space refers to, for example, a first chamber, a liquid reservoir, and space α. A culture vessel according to one embodiment of the present invention preferably has a second chamber as part of the culture space. Alternatively, another space may be provided as part of the culture space, with one end communicating with the first or second chamber and the other end communicating with space α. The culture space is preferably a space that allows the culture medium or culture medium necessary for culturing the cultured organism to come into contact with the cultured organism, or that can enclose them.

[0118] [Space α] In one embodiment of the present invention, space α is a part of the culture space. Space α is suitable for three-dimensional control of cell growth and the like. In one embodiment of the present invention, it is preferable that the space α in the culture vessel is a flow channel.

[0119] [Flow path] In one embodiment of the present invention, the flow path is a tubular space in which a cultured organism can grow in a directional manner, and which is not closed at both ends so that substances such as culture medium necessary for cultivation can flow through it. The state in which both ends are not closed means that the flow path is not blocked by a solid such as resin, and is connected to some space in which liquids and gases can be supplied to space α. Both ends of the flow path are open to the space outside the container, such as the atmosphere, or are connected to some other void formed by the first chamber, second chamber, liquid reservoir, or other constituent members.

[0120] The two ends of the flow path may be connected to different locations. For example, one end may be connected to the first chamber and the other end to the second chamber.

[0121] In one embodiment of the present invention, the culture vessel preferably has a channel width in the range of 1 μm to 1000 μm and a depth in the range of 1 μm to 1000 μm, more preferably has a channel width in the range of 10 μm to 1000 μm and a depth in the range of 10 μm to 1000 μm, more preferably has a channel width in the range of 10 μm to 200 μm and a depth in the range of 10 μm to 200 μm, more preferably has a channel width in the range of 20 μm to 200 μm and a depth in the range of 20 μm to 200 μm, more preferably has a channel width in the range of 30 μm to 180 μm and a depth in the range of 30 μm to 180 μm, and even more preferably has a channel width in the range of 50 μm to 100 μm and a depth in the range of 50 μm to 100 μm.

[0122] In one embodiment of the present invention, the culture vessel preferably has a channel length in the range of 1 mm to 100 mm, more preferably in the range of 1 mm to 50 mm, more preferably in the range of 5 mm to 30 mm, more preferably in the range of 5 mm to 20 mm, and even more preferably in the range of 5 mm to 15 mm.

[0123] In one embodiment of the present invention, it is preferable to use space α as a microfluidic channel in the culture vessel. A culture vessel having a microfluidic channel is also called a microfluidic device or microfluidic chip. Microfluidic devices are a general term for devices that incorporate microfluidic channels and reaction vessels by microfabrication of the culture surface of a culture vessel, and are applied to bio-research and chemical engineering. Examples of microfluidic devices include those called microTAS (micro Total Analysis Systems) and Lab on a Chip. These devices are being increasingly applied as next-generation culture equipment.

[0124] [Cultivated material] A culture vessel according to one aspect of the present invention has a culture space for culturing a substance to be cultured. The cultured material is a cell, tissue, or organ. In one embodiment of the present invention, cells, tissues, or organs are also referred to simply as "cells, etc." The origin of the cells, etc., is not particularly limited and may be any living organism such as animals, plants, insects, fungi, protists, bacteria, etc., but animals or plants are preferred, animals are more preferred, and mammals are even more preferred.

[0125] In one embodiment of the present invention, a culture vessel is provided with sufficient oxygen necessary for cell growth from the bottom of the space α in which cells are cultured. Furthermore, since space α is suitable for three-dimensional control of cell growth, and the cultured cells can be removed without damage by peeling off the film member (A) and container member (B) after culture, it is preferable that the cultured cells are cells. In one embodiment of the present invention, the cells, etc., are not particularly limited, but are preferably aerobic, and more preferably do not contain anaerobic cells, etc. In one embodiment of the present invention, the cells may be floating cells or adherent cells, but are preferably adherent cells.

[0126] Examples of adherent cells include fibroblasts, mesenchymal stem cells, hematopoietic stem cells, neural stem cells, nerve cells, corneal epithelial cells, oral mucosal cells, retinal pigment cells, periodontal ligament stem cells, myofibroblasts, cardiomyocytes, hepatocytes, splenic endocrine cells, keratinocytes, dermal fibroblasts, subcutaneous adipose-derived progenitor cells, kidney cells, basal hair root sheath cells, nasal mucosal epithelial cells, vascular endothelial progenitor cells, vascular endothelial cells, vascular smooth muscle cells, osteoblasts, chondrocytes, skeletal muscle cells, immortalized cells, cancer cells, keratinocytes, embryonic stem cells (ES cells), EBV-transformed B cells, and induced pluripotent stem cells (iPS cells).

[0127] The cells may be either primary cultured cells or cell lines that have been passaged, but primary cultured cells are preferred. Frozen or refrozen cells may also be used. The cells may be cells cultured in two dimensions or cells cultured in three dimensions. The cells include spheroids obtained by culturing cells. Preferably, the cells are cells cultured in three dimensions.

[0128] The cells are preferably those with high oxygen requirements, such as cells that make up skin, kidney, liver, brain, nerve tissue, myocardial tissue, skeletal muscle tissue, cancer stem cells, and cancer cells. Among these, cells that make up skin, kidney, liver, brain, nerve tissue, myocardial tissue, or skeletal muscle tissue, or cancer cells, are preferred because they have high oxygen requirements. In one embodiment of the present invention, the cultured organism is more preferably nerve cells or cardiomyocytes, and even more preferably nerve cells. The presence of nerve cells is preferable because it allows for sufficient oxygen supply during culture and is suitable for three-dimensional control of nerve cell axonal bundle growth.

[0129] In one embodiment of the present invention, the culture vessel is preferably configured such that the first through-hole is a first chamber for receiving the cultured material, the cell bodies of nerve cells are received in the first chamber, and the axonal bundles extending from the cell bodies are received in space α.

[0130] In one embodiment of the present invention, "tissue" means a collection of similar cells that perform similar functions. The tissue is not particularly limited and includes, for example, epithelial tissue, connective tissue, muscle tissue, nerve tissue, and the like. In one aspect of the present invention, an organ means a collection of tissues that work together to perform a purposeful collaborative task. The organ is not particularly limited and may include, for example, the lungs, heart, liver, kidneys, spleen, pancreas, gallbladder, esophagus, stomach, skin, brain, etc. The cultured organisms may be cultured individually or in combination of two or more organisms.

[0131] In one embodiment of the present invention, "culture" is used in a broad sense to include not only the proliferation and maintenance of cells, but also processes such as seeding, subculturing, differentiation induction, and self-organization induction of cells. The culture medium used for culture is not limited, and a medium can be selected according to the characteristics of the cells.

[0132] <Culture method> A culture method according to one aspect of the present invention comprises a step (I) of incubating cells, tissues, or organs in a culture vessel having a container member (B) having an inverted recess on the lower surface of the bottom and a first through hole at one end of the inverted recess, and an oxygen-permeable film member (A), wherein the film member (A) and the container member (B) are detachably attached to each other, and in the state in which the film member (A) and the container member (B) are attached, a space α is formed by the inverted recess and the film member (A), the first through hole located at one end of the space α receives the cultured material, and the space α is part of the culture space. In a culture method according to one aspect of the present invention, the cells, tissue, or organ is more preferably a nerve cell or a cardiomyocyte, and even more preferably a nerve cell. One embodiment of the present invention is suitable for three-dimensional control of the growth of axonal bundles of nerve cells, and allows for the removal of axonal bundles without damage; therefore, it is preferable that the cells, tissues, or organs are nerve cells.

[0133] A culture method according to one aspect of the present invention preferably includes, after step (I) above, a peeling step (II) in which the film member (A) and the container member (B) are separated, and after the peeling step (II), a removal step (III) in which the incubated cells, tissue, or organs are removed. In the peeling step (II), it is preferable to keep the film member (A) horizontal in order to remove the incubated cells etc. without damage.

[0134] <Method for manufacturing culture vessels> A method for manufacturing a culture vessel according to one aspect of the present invention comprises a bonding step of peelably bonding a container member (B) having an inverted recess on the underside of the bottom and a first through hole at one end of the inverted recess, to an oxygen-permeable film member (A). According to a manufacturing method in one aspect of the present invention, after the above bonding step, when the film member (A) and the container member (B) are bonded together, a space α is formed by the inverted recess and the film member (A), the first through-hole located at one end of the space α receives the cultured material, and the space α is part of the culture space, thereby enabling the manufacture of a culture container.

[0135] (Application process) In a method for manufacturing a culture vessel according to one aspect of the present invention, the adhesion step is not particularly limited, but can be carried out, for example, by using a heat press and laser irradiation. In one embodiment of the present invention, the bonding step is preferably performed using a hot press because it is easy to bond the bonded parts together in a peelable manner. When the bonding process is performed by hot pressing, a hot press molding machine is used. There are no limitations on the hot pressing conditions, but it is preferable to heat at 20 to 200°C and press for 1 second to 10 minutes, and more preferably to heat at 50 to 150°C and press for 5 seconds to 1 minute.

[0136] In a method for manufacturing a culture vessel according to one aspect of the present invention, the bonding step preferably includes either a step of hydrophilizing part or all of the bonding surfaces of the film member (A) and the container member (B), followed by a step of heat-pressing the hydrophilized bonding surfaces, or a laser irradiation step of irradiating the bonding surface of the film member (A) and the bonding surface of the container member (B) with a laser while they are in contact.

[0137] In one embodiment of the present invention, the adhesive surface refers to the surface to which the film member (A) and the container member (B) are adhered. More specifically, in the film member (A), "part of the adhesive surface" refers to the surface that constitutes space α, which is the upper surface of the film member (A). Furthermore, "the entire adhesive surface" refers to the upper surface of the film member (A). In container member (B), "part of the adhesive surface" refers to the surface that constitutes the space α, which is an inverted recess on the bottom surface of container member (B). "The entire adhesive surface" refers to the bottom surface of container member (B).

[0138] In one embodiment of the present invention, the bonding process for a culture container is more preferably characterized by hydrophilizing the entire bonding surface of the film member (A) by vacuum plasma treatment, hydrophilizing the entire bonding surface of the container member (B) by atmospheric pressure plasma treatment, and then heat-pressing the hydrophilized bonding surfaces. [Examples]

[0139] One aspect of the present invention will be described in more detail below based on examples, but the present invention is not limited to these examples.

[0140] (measurement) The methods for measuring the oxygen permeability coefficient, calculating oxygen permeability, measuring peel strength, measuring total light transmittance, measuring the glass transition temperature, and measuring the water contact angle in the examples are described below.

[0141] [Method for measuring oxygen permeability coefficient and method for calculating oxygen permeability] For oxygen permeability, the measurement was conducted in accordance with the Japanese Industrial Standard JIS-K-7126-1. The oxygen permeability coefficient of the measurement sample was measured using a differential pressure gas permeability measuring device MT-C3 manufactured by Toyo Seiki Seisakusho under conditions of 23°C and 0% humidity. The measurement section diameter was 70 mm (permeability area was 38.46 cm²). 2 ) was used. Since a large oxygen permeability coefficient was expected, an aluminum mask was applied to the sample beforehand, and the actual permeability area was set to 5.0 cm². 2 That's what I decided. Measured oxygen permeability coefficient [cm 3 ×mm / (m 2 The value of (×24h×atm) is divided by the thickness (μm) of the film member (A) or coverslip to obtain the oxygen permeability [cm 3 / (m 2 The formula (×24h×atm) was calculated.

[0142] [Measurement of peel strength] Regarding peel strength, it was measured as 90° peel strength using a tensile testing machine (SVZ-500NB model, manufactured by Imada Seisakusho) in accordance with Japanese Industrial Standard JIS-K6854-1:1999 (Adhesives - Test method for peel strength, 90° peel).

[0143] [Measurement of total light transmittance] For the measurement samples, the total light transmittance was measured using a haze meter (NDH2000, manufactured by Nippon Denshoku Industries Co., Ltd.) in accordance with the Japanese Industrial Standard JIS-K-7361-1.

[0144] [Measurement of glass transition temperature] The glass transition temperature was measured using a differential calorimeter in accordance with Japanese Industrial Standard JIS-K7121:1987 (Method for measuring transition temperature of plastics).

[0145] [Measuring the water contact angle] The water contact angle after hydrophilization treatment of the culture surface was measured in accordance with Japanese Industrial Standard JIS-R3257 (Test method for wettability of substrate glass surfaces). Under constant temperature and humidity conditions of 25±5℃ and 50±10%, a water droplet of 4 μL or less, whose shape can be considered spherical, was dropped onto the surface of the sample to be measured. The angle (°) of the contact interface between the substrate and the water droplet was measured using the static drop method, from immediately after the water droplet contacted the sample surface to within 1 minute. In this embodiment, the values ​​obtained within one minute immediately after contact with the water droplet using the method described above were treated as physical property values.

[0146] [Manufacturing Example 1] Manufacturing of films (A-1) and (A-2) 4-methyl-1-pentene polymer (trade name: TPX, manufactured by Mitsui Chemicals, Inc., total light transmittance 92%, glass transition temperature 40℃, weight-average molecular weight (Mw) 42.8 × 10⁻⁶) 4Using a 3D printer, the material was fed into an extruder equipped with a T-die and a full-flight screw to extrude the substrate layer. The extrusion temperature was set to 270°C, the roll temperature to 60°C, and the roll rotation speed was varied during the extrusion process to obtain two films of different thicknesses. The 50 μm thick film was designated as film (A-1), and the 100 μm thick film was designated as film (A-2). Film (A-1) and film (A-2) were used as film member (A) in the examples. A coverslip was used as a comparative example instead of film member (A).

[0147] Film (A-1): A 4-methyl-1-pentene polymer film with a thickness of 50 μm was used. The 4-methyl-1-pentene polymer (X) content in 100% by mass of film (A-1) was 99.5% by mass. When the total amount of repeating constituent units of 4-methyl-1-pentene polymer (X) was set to 100 mol%, the content of constituent units derived from 4-methyl-1-pentene was 98.4 mol%. Film (A-2): A 4-methyl-1-pentene polymer film with a thickness of 100 μm was used. The 4-methyl-1-pentene polymer (X) content in 100% by mass of film (A-1) was 99.5% by mass. When the total amount of repeating constituent units of 4-methyl-1-pentene polymer (X) was set to 100 mol%, the content of constituent units derived from 4-methyl-1-pentene was 98.4 mol%.

[0148] Coverslip: A borosilicate glass slide with a thickness of 1000 μm (manufactured by Matsunami Glass Industry Co., Ltd., product name: Frost Slide Glass S2112) was used.

[0149] Material, thickness (μm), and oxygen permeability coefficient (cm) of film (A-1), film (A-2), and coverslip. 3 · mm / m 2 (24h·atm), oxygen permeability (cm²) 3 / m 2 Table 1 shows the (24h·atm) values.

[0150] [Table 1]

[0151] [Manufacturing Example 2] Manufacturing of container components (B-1) and (B-2) Container component (B) was manufactured using injection molding with 4-methyl-1-pentene polymer (product name: TPX, manufactured by Mitsui Chemicals, Inc., total light transmittance 92%, glass transition temperature 30°C) or polystyrene (product name: Toyo Styrofoam GP MW1D, manufactured by Toyo Styrene Co., Ltd., total light transmittance 90%, glass transition temperature 100°C) as raw materials. Container component (B-1) was manufactured using 4-methyl-1-pentene polymer as a raw material, and container component (B-2) was manufactured using polystyrene as a raw material.

[0152] The shapes of the container members (B-1) and (B-2) are as shown in Figure 1. The top surface of container member (B) is open, and the bottom surface has two cylindrical through holes, one with a diameter of 1 mm and the other with a diameter of 0.5 mm. In the culture test described later, the first cylindrical through hole with a diameter of 1 mm was used as the first chamber, and the second cylindrical through hole with a diameter of 0.5 mm was used as the second chamber. The dimensions of the container components (B-1) and (B-2) were 2.4 cm in length, 1.2 cm in width, and 1.1 cm in height.

[0153] On the bottom surface, there were two grooves with an inverted U-shape cross-section that connected one end of the first through-hole and one end of the second through-hole. The inverted U-shaped grooves had a semicircular cross-section, a diameter of 80 μm, and a length of 15 mm. These grooves become space α and flow channels when film (A-1) or (A-2) is attached in a later process. There were two of the aforementioned spaces α on the bottom surface of the container members (B-1) and (B-2). The volume of the liquid reservoirs in container members (B-1) and (B-2) was 2.3 mL.

[0154] As a comparative example, a container component made of polydimethylsiloxane (P-Chip manufactured by Jiksak Bioengineering Co., Ltd.) was prepared and designated as the container component (comparative example). The container component (comparative example) has the same shape as container components (B-1) and (B-2).

[0155] [Manufacturing Example 3] Manufacturing of culture vessels (vacuum plasma treatment) The entirety of the film (A-1), container component (B-1), and container component (B-2) was subjected to a hydrophilic treatment. Vacuum plasma treatment was performed as the hydrophilic treatment. Table 2 shows the water contact angle (°) before and after vacuum plasma treatment.

[0156] [Table 2]

[0157] Film (A-1), container member (B-1), and container member (B-2), which had been treated with vacuum plasma, were used to manufacture culture vessels 1 to 4. Film (A-1) was attached to the bottom surface of container member (B-1) and container member (B-2) respectively by hot pressing. The hot pressing conditions for culture vessels 1 to 4 are shown in Table 3.

[0158] [Table 3]

[0159] [Manufacturing Example 4] Manufacturing of culture vessels (atmospheric pressure plasma treatment) Both film (A-1) and film (A-2) were subjected to hydrophilic treatment using an atmospheric pressure plasma surface treatment device (manufactured by Sekisui Chemical Co., Ltd.). For atmospheric pressure plasma treatment, corona treatment was performed using a table-type corona treatment device (manufactured by Kasuga Electric Co., Ltd.) (processing speed 3 m / min, output 0.5 kW, 2 passes). Table 4 shows the water contact angles (°) of films (A-1) and (A-2) that have undergone atmospheric pressure plasma treatment.

[0160] [Table 4]

[0161] Culture vessels 5 and 6 were manufactured by attaching either film (A-1) or film (A-2), which had been subjected to atmospheric pressure plasma treatment, to the bottom surface of container member (B-1) by irradiation with a carbon dioxide laser beam. Note that container member (B-1) was not subjected to atmospheric pressure plasma treatment.

[0162] Culture vessels 5 and 6 were prepared by irradiating them with a carbon dioxide laser beam using a carbon dioxide laser (wavelength = 10.6 μm, continuous wave oscillation, beam diameter = approximately 2 mm) by attaching a film member (A) and a container member (B), and irradiating them from the film side with a carbon dioxide laser output of 0.03 J / mm and a feed rate of 200 mm / second.

[0163] As a comparative example, a culture container 7 was manufactured by attaching a coverslip that had not been treated to hydrophilicity to the bottom surface of a container component (comparative example) using a commercially available sealing material.

[0164] [Example 1] Measurement of peel strength and presence or absence of damage The peel strength of culture vessels 1 to 7 was measured using the measurement method described above. After measuring the peel strength, the film and container components were observed for any damage. The results for culture containers 1-6 as examples and culture container 7 as a comparative example are shown in Table 5.

[0165] [Example 2] Leakage test Ethanol was filled into the liquid reservoirs of culture vessels 1 to 7 and left for 12 hours. After that, the presence or absence of liquid leakage was visually checked at the adhesive surface between the film member and the container member. The results for culture vessels 1 to 6 as examples and culture vessel 7 as a comparative example are shown in Table 5.

[0166] [Table 5]

[0167] [Example 3] Culture test (Motor neuron spheroid production) Spheroid 1 was produced by seeding human iPS cell-derived motor neurons (Elixirgen Scientific) onto commercially available culture plates and culturing them at 37°C for 7 days in an atmosphere of 20% oxygen, 5% carbon dioxide, and 75% nitrogen.

[0168] (Sterilization of containers) Ethanol was filled into the liquid reservoirs of culture vessels 1-7. The fact that space α was filled with ethanol was confirmed by visual observation through the film or coverslip attached to the bottom surface of the culture vessels. Culture vessels 1-7, filled with ethanol, were left to stand at room temperature for 10 minutes. After that, all the ethanol in the culture vessels was drained through the opening on the top of the liquid reservoir, air-dried at room temperature, and then sterilized with ultraviolet light.

[0169] (Coating treatment of the culture surface) Geltrex (manufactured by Thermo Fisher Scientific) was dissolved in sterile water to prepare a coating solution. The coating solution was filled into the liquid reservoirs of sterile culture vessels 1-7. The fact that space α was filled with the coating solution was confirmed by visual observation through the film or coverslip attached to the bottom surface of the culture vessels. After standing at 37°C for 1 hour, the culture surface was coated by draining all of the coating aqueous solution from the culture vessel through the opening on the top of the liquid reservoir.

[0170] (Preparation of axon bundles) After seeding nerve cells (spheroid 1) in the first chamber at the bottom of culture vessels 1-7, which had undergone surface treatment, the liquid reservoir of each vessel was filled with culture medium (Thermo Fisher, product name: Neurobasal Plus Medium). The fact that space α was filled with culture medium was confirmed by visual observation through the film or coverslip attached to the bottom surface of the culture vessel.

[0171] The cultures were incubated at 37°C for 17 days in an atmosphere of 20% oxygen, 5% carbon dioxide, and 75% nitrogen. Visual inspection was conducted through a film or coverslip from the bottom surface of each of the culture vessels (1-7) to confirm that axons were growing in the channel, which is space α. After culturing axon bundles in the channels of culture vessels 1 to 7, all the culture medium in the culture vessel was drained from the opening on the top surface of the liquid reservoir to obtain a culture vessel containing nerve cells with axon bundles.

[0172] (Extraction of axon bundles) For culture vessels 1 to 6 in the example, the film at the bottom of the vessel was peeled off by hand from the culture vessel containing nerve cells with axon bundles, and nerve cells containing axon bundles that had grown in the channel were collected with tweezers.

[0173] In the case of culture vessel 7 of the comparative example, since the coverslip at the bottom of the vessel could not be peeled off by hand, sterile water was injected under pressure from the second chamber using a pipette, and the axon bundles that had grown in the flow path were discharged from the first chamber side along with the sterile water to remove them, and the nerve cells containing the axon bundles were collected with tweezers.

[0174] (Evaluation of nerve cells after culture) Nerve cells containing axon bundles (spheroid 1) collected from culture vessels 1-7 were observed using a light microscope. Specifically, observation was performed using a light microscope through a film attached to the bottom surface of the culture vessels. The observation results for culture vessels 1, 3, and 4 are shown in Figure 3. When cultured in culture vessels 1, 3, and 4, the axon bundles grew to lengths of 15 mm, 15 mm, and 10 mm, respectively. The nerve cells cultured in culture vessels 1-6 were cultured in a morphology similar to that of living tissues by controlling their growth in three dimensions.

[0175] Although the growth of nerve cells cultured in culture vessel 7 was controlled in three dimensions, the axons became distorted and aneurysms formed, and the cells were not cultured in a morphology similar to that of tissue in vivo.

[0176] (Assessment of whether or not there is damage to the axon bundle) Nerve cells containing axonal bundles (spheroid 1) collected from culture vessels 1, 3, 5, and 7 were observed under a light microscope to check for damage to the axonal bundles. Cases where the axon bundle was severed but showed little to no damage such as breakage were evaluated as "no damage." Cases where the axon bundle was severed and damage such as breakage was clearly observed were evaluated as "damaged." Table 6 shows the results of observing nerve cells containing axon bundles (spheroid 1) collected from each of the culture vessels 1, 3, 5, and 7, which were cultured 10 times under the same conditions.

[0177] [Table 6]

[0178] These results revealed that in culture vessels 1, 3, and 5, the film could be peeled off and the axon bundles removed without damage, but in culture vessel 7, the coverslip could not be peeled off, resulting in damage to a certain percentage of the axon bundles.

[0179] Culture vessels 1-6 provided sufficient oxygen for growth from the bottom of the culture vessel where nerve cells adhered, and had a culture space suitable for three-dimensional control of nerve cell growth. As a result, nerve cells could be cultured in a form similar to that of tissue in vivo. Furthermore, the axonal bundles of nerve cells cultured in culture vessels 1, 3, and 5 could be removed without damage by peeling the film member (A) from the container member (B).

[0180] On the other hand, in culture vessel 7, insufficient oxygen was supplied to the bottom of the culture vessel where the nerve cells adhered, causing the axons to become distorted and aneurysms to form. As a result, it was not possible to culture nerve cells in a morphology similar to that of living tissue. Furthermore, the axon bundles of nerve cells cultured in culture vessel 7 were damaged, such as being cut or broken, because the film member (A) could not be peeled off from the container member (B), and sterile water was repeatedly injected into the first chamber to remove them. [Explanation of Symbols]

[0181] 1: Film component (A) 2: Container component (B) 10:First through hole 11: First Chamber Section 20:Second through hole 21: Second Chamber Section 30: Liquid reservoir 40: Reverse concave 50: Space α 60: Cell body of nerve cell (spheroid 1) 70: Axon bundle extending from the cell body of a nerve cell (spheroid 1)

Claims

1. A culture vessel having a culture space for culturing a subject, The culture vessel is A container member (B) having a liquid reservoir for containing a culture medium, an inverted recess on the lower surface of the bottom of the liquid reservoir, and a first through hole at one end of the inverted recess and a second through hole at the other end, The container member (B) has an oxygen-permeable film member (A) that is peelably attached to the lower surface of the container member (B), In the state in which the film member (A) and the container member (B) are attached to each other, a flow path is formed by the inverted recess and the film member (A). The first through-hole located at one end of the channel receives the cultured material, The channel is part of the culture space, A culture vessel in which the flow path and the liquid reservoir are in communication via the first through-hole and the second through-hole.

2. The culture vessel according to claim 1, wherein the width of the channel is in the range of 1 μm to 1000 μm and the depth is in the range of 1 μm to 1000 μm.

3. The culture vessel according to claim 1 or 2, wherein the length of the flow channel is in the range of 1 mm to 100 mm.

4. The culture vessel according to claim 1 or 2, wherein the thickness of the film member (A) is in the range of 1 μm to 1000 μm.

5. The culture vessel according to claim 1 or 2, wherein the inverted recess is an inverted recess for flow channels.

6. The culture container according to claim 1 or 2, wherein the peel strength in a 90° peel test between the film member (A) and the container member (B), measured in accordance with JIS-K6854-1:1999, is in the range of 0.01 to 3.0 N / cm.

7. The culture vessel according to claim 1 or 2, wherein the total light transmittance of the film member (A), as measured in accordance with JIS-K-7361-1, is 80% or more.

8. The oxygen permeability of the film member (A) at a temperature of 23°C and humidity of 0% is 4500 to 90000 cm². ３ / (m ２ A culture vessel according to claim 1 or 2, wherein the temperature is within the range of ×24h × atm.

9. The culture container according to claim 1 or 2, wherein the container member (B) includes resin.

10. The culture vessel according to claim 9, wherein the resin is a thermoplastic resin.

11. The culture vessel according to claim 10, wherein the glass transition temperature of the thermoplastic resin, as measured in accordance with JIS-K7121:1987, is 120°C or lower.

12. The culture vessel according to claim 1 or 2, wherein the film member (A) comprises a 4-methyl-1-pentene polymer (X).

13. The culture container according to claim 1 or 2, wherein the container member (B) contains a 4-methyl-1-pentene polymer (X).

14. The culture vessel according to claim 12, wherein the 4-methyl-1-pentene polymer (X) is at least one polymer selected from a 4-methyl-1-pentene homopolymer (X1) and a copolymer (X2) of 4-methyl-1-pentene and at least one olefin selected from ethylene, propylene, and α-olefins having 4 to 20 carbon atoms.

15. The culture container according to claim 1 or 2, wherein the water contact angle of at least the culture surface of either the film member (A) or the container member (B) is 50° to 100°.

16. The culture container according to claim 1 or 2, wherein at least one culture surface of either the film member (A) or the container member (B) is a culture surface coated with at least one material selected from the group consisting of natural polymer materials, synthetic polymer materials, and inorganic materials.

17. The culture container according to claim 1 or 2, wherein at least one of the culture surfaces of the film member (A) and the container member (B) is a culture surface that has been treated to be hydrophilic.

18. The culture vessel according to claim 1 or 2, wherein the cultured material is nerve cells or cardiomyocytes.

19. The first through-hole is the first chamber portion that receives the cultured material, The culture vessel according to claim 18, wherein the cell bodies of the nerve cells are received in the first chamber, and the axonal bundles extending from the cell bodies are received in the channel.

20. A method for culturing cells, tissues, or organs, comprising the step (I) of incubating cells, tissues, or organs in a culture vessel according to claim 1 or 2, by placing a culture medium in the liquid reservoir.

21. After step (I), a peeling step (II) is performed to separate the film member (A) and the container member (B). The culture method according to claim 20, further comprising a removal step (III) of removing incubated cells, tissue, or organs after the detachment step (II).

22. A method for manufacturing a culture vessel according to claim 1 or 2, A container member (B) having a liquid reservoir for containing a culture medium, an inverted recess on the lower surface of the bottom of the liquid reservoir, and a first through hole at one end of the inverted recess and a second through hole at the other end, The process includes a bonding step of peelably bonding an oxygen-permeable film member (A) to the lower surface of the container member (B), The inverted recess and the film member (A) form a flow path. A method for manufacturing a culture vessel, wherein the flow path and the liquid reservoir are in communication via the first through-hole and the second through-hole.

23. The pasting step includes: A step of treating part or all of the adhesive surfaces of the film member (A) and the container member (B) to make them hydrophilic, and then heat-pressing the hydrophilic treated adhesive surfaces. or Laser irradiation process: Laser irradiation is performed while the adhesive surface of the film member (A) and the adhesive surface of the container member (B) are in contact. A method for producing a culture vessel according to claim 22, comprising any of the following:

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