Gas-permeable film and production method therefor, and culture container, biomimetic system including same, and observation method

Abstract

The present invention addresses the problem of providing a gas-permeable film which has gas permeability and can be used in a culture container, and through which cells and the like can be observed. The gas-permeable film for solving the problem contains a 4-methyl-1-pentene-based polymer. At least one surface of the gas-permeable film has a root mean square slope (Sdq) of 0.0000-0.0100 as measured in accordance with ISO 25178-2:2012, a developed area ratio (Sdr) of 0.0000-0.0070 as measured in accordance with ISO 25178-2:2012, and an oxygen permeability at 23°C of 1.0-100.0 L / (m2･24 h･atm) as measured in accordance with JIS K7126-1:2006.<sp / >

Description

Gas permeable film and method for producing the same, as well as a culture vessel, a biomimetic system including the same, and observation method.

[0001] The present invention relates to a gas-permeable film and a method for producing the same, as well as a culture vessel, a biomimetic system including the same, and an observation method.

[0002] Cells, tissues, organs, and microorganisms cannot be cultured unless the conditions are suitable for their growth. Therefore, when culturing, it is common practice to place cells and a culture medium containing appropriate nutrients into culture vessels such as dishes, plates, or flasks, and then place these culture vessels in an incubator maintained at a predetermined temperature, humidity, and gas concentration. In this process, various gas-permeable materials are being considered for the culture vessels in order to properly supply oxygen and carbon dioxide. For example, the application of polyethylene resin and polypropylene resin is being considered (Patent Document 1, etc.).

[0003] Japanese Patent Publication No. 2022-149254

[0004] However, polyethylene and polypropylene resins have a drawback: increasing their thickness to ensure sufficient strength often compromises their oxygen permeability. Furthermore, culture vessels are required to allow observation and optical detection of cells and other organisms cultured within them without removing them. However, polyethylene and polypropylene resins tend to lose transparency when thickened. Therefore, observing and detecting cells and other organisms within the culture vessel can be difficult.

[0005] This invention has been made in view of the above problems. The present invention aims to provide a gas-permeable film that can be used in culture vessels and is suitable for observation and detection of cells and the like. Furthermore, the present invention aims to provide a method for manufacturing the above gas-permeable film, a culture vessel using the above gas-permeable film, and a biomimetic system and observation method including the same.

[0006] The present invention provides the following gas permeable film: [1] A gas permeable film comprising a 4-methyl-1-pentene polymer, wherein the root mean square slope (Sdq) of at least one surface, measured in accordance with ISO 25178-2:2012, is 0.0000 or more and 0.0100 or less, and the unfolded area ratio (Sdr), measured in accordance with ISO 25178-2:2012, is 0.0000 or more and 0.0070 or less, and the oxygen permeability at a temperature of 23°C and humidity of 0%, measured in accordance with JIS K7126-1:2006, is 1.0 L / (m²). ２ ・24h・atm) or more 100.0L / (m ２ [1] A gas permeable film having a humidity of 24 h·atm or less. [2] The gas permeable film according to [1], wherein the 4-methyl-1-pentene polymer comprises constituent units derived from 4-methyl-1-pentene and constituent units derived from ethylene and / or α-olefins having 3 to 20 carbon atoms (excluding 4-methyl-1-pentene). [3] The gas permeable film according to [1] or [2], wherein when the total constituent units of the 4-methyl-1-pentene polymer are set to 100 mol%, the amount of constituent units derived from 4-methyl-1-pentene is 60 mol% or more and 99 mol% or less. [4] The gas permeable film according to any one of [1] to [3], wherein the thickness is 10 μm or more and 500 μm or less. [5] The gas permeable film according to any one of [1] to [4], wherein the standard deviation of brightness is 0.0 or more and 4.0 or less. [6] A gas permeable film according to any of [1] to [5], wherein the haze measured in accordance with JIS K7136:2000 is 0.01% or more and 3.00% or less. [7] A gas permeable film according to any of [1] to [6], wherein the Rockwell hardness measured on the R scale in accordance with ISO 2039-2:1987 is 80 or more and 115 or less.

[0007] The present invention provides the following methods for manufacturing a gas permeable film: [8] A method for manufacturing a gas permeable film, comprising the steps of: preparing a resin composition for film molding containing a 4-methyl-1-pentene polymer; and forming the molten resin composition for film molding into a film by passing it between two opposing rolls, wherein the root mean square slope (Sdq) of the surface of at least one of the two opposing rolls, measured in accordance with ISO 25178-2:2012, is 0.0000 or more and 0.0030 or less, and the unfolded area ratio (Sdr), measured in accordance with ISO 25178-2:2012, is 0.0000 or more and 0.0007 or less. [9] The method for manufacturing a gas permeable film according to [8], wherein the melt flow rate of the 4-methyl-1-pentene polymer at 260°C under a 5.0 kg load is 5 g / 10 min or more and 180 g / 10 min or less.

[10] A method for manufacturing a gas permeable film, comprising the steps of: preparing a film containing a 4-methyl-1-pentene polymer; and sandwiching the film between two plate-like members and pressing it, wherein the root mean square slope (Sdq) of at least one of the two plate-like members facing the film, measured in accordance with ISO 25178-2:2012, is 0.0000 or more and 0.0030 or less, and the unfolded area ratio (Sdr), measured in accordance with ISO 25178-2:2012, is 0.0000 or more and 0.0007 or less.

[0008] The present invention provides the following culture vessels, biomimetic systems, and observation methods:

[11] A culture vessel using a gas permeable film as described in any of [1] to [7] above.

[12] A culture vessel comprising a main body having through holes and the gas permeable film attached to the main body so as to close one end of the through holes.

[13] A biomimetic system including the culture vessel of

[11] above.

[14] An observation method comprising the steps of culturing cells or microorganisms in a containment section surrounded by the wall surface of the through holes and the gas permeable film of the culture vessel of

[12] above, and observing the cells or microorganisms.

[0009] The present invention provides a gas-permeable film that can be used in cell culture vessels and is suitable for observation and detection of cells and other elements.

[0010] Figure 1 is a perspective view of the culture vessel. Figure 2 is a cross-sectional view of a part of the culture vessel. Figure 3A is a photograph under the insert when co-culturing was performed using the gas permeable film of Example 1, and Figure 3B is a photograph under the insert when co-culturing was performed using a polystyrene film.

[0011] 1. Gas Permeable Film The gas permeable film of the present invention will be described using one embodiment as an example. However, the gas permeable film of the present invention is not limited to the following embodiment.

[0012] The gas permeable film of this embodiment is a film containing a 4-methyl-1-pentene polymer. The gas permeable film may consist only of the 4-methyl-1-pentene polymer, or it may contain the 4-methyl-1-pentene polymer and other components. However, the content of the 4-methyl-1-pentene polymer is preferably 80% by mass, more preferably 85% by mass or more, and particularly preferably 90% by mass or more, relative to the total mass of the gas permeable film. In particular, when the amount of the 4-methyl-1-pentene polymer relative to the total mass of the gas permeable film is 95% by mass or more, the rigidity of the gas permeable film is increased, making it less prone to deformation such as bending when used as a component of a cell culture vessel, further improving the observationability during cell observation, and increasing the surface hardness of the gas permeable film, improving scratch resistance.

[0013] In this specification, a 4-methyl-1-pentene polymer is a polymer containing structural units derived from 4-methylpentene, and may be a homopolymer of 4-methyl-1-pentene, or a copolymer of 4-methyl-1-pentene with one or more other monomers. The amount of structural units derived from 4-methyl-1-pentene relative to the total structural units of the 4-methyl-1-pentene polymer may be 50 mol% or more, preferably 60 mol% to 99 mol%, and more preferably 80 mol% to 99 mol%.

[0014] The type of monomer copolymerizable with 4-methyl-1-pentene is not particularly limited as long as it does not impair the purpose and effects of this embodiment, but ethylene and / or α-olefins having 3 to 20 carbon atoms (excluding 4-methyl-1-pentene) are preferred. When the 4-methyl-1-pentene polymer is a copolymer of 4-methyl-1-pentene with ethylene or an α-olefin, the melting point and softening point of the 4-methyl-1-pentene polymer tend to fall within the desired range. As a result, it is easier to manufacture by the manufacturing method described later, and the root mean square slope (Sdq (hereinafter also simply referred to as "Sdq")) and the developed area ratio (Sdr (hereinafter also simply referred to as "Sdr")) of the surface of the gas permeable film tend to fall within the desired range.

[0015] The above α-olefins may be linear, branched, or have a cyclic structure. Examples of linear α-olefins include propylene, 1-butene, 1-pentene, 1-hexene, 1-octene, 1-decene, 1-undecene, 1-dodecene, 1-tetradecene, 1-hexadecene, 1-octadecene, and 1-eicosene. Examples of branched α-olefins include 3-methyl-1-butene, 3-methyl-1-pentene, 3-ethyl-1-pentene, 4,4-dimethyl-1-pentene, 4-methyl-1-hexene, 4,4-dimethyl-1-hexene, 4-ethyl-1-hexene, and 3-ethyl-1-hexene. Examples of α-olefins having a cyclic structure include cyclopentene, cyclohexene, cycloheptene, norbornene, 5-methyl-2-norbornene, tetracyclododecene, and vinylcyclohexane. The 4-methyl-1-pentene polymer is preferably a copolymer of 4-methyl-1-pentene with 1-octene, 1-decene, 1-dodecene, 1-tetradecene, 1-hexadecene, 1-heptadecene, and 1-octadecene, and a copolymer of 4-methyl-1-pentene and 1-decene is more preferable from the viewpoint of easily increasing the rigidity and hardness of the gas permeable film.

[0016] Furthermore, the melt flow rate (MFR) of the 4-methyl-1-pentene polymer at a 5.0 kg load is preferably 5 g / 10 min to 180 g / 10 min, and more preferably 10 g / 10 min to 100 g / 10 min. When the melt flow rate of the 4-methyl-1-pentene polymer is within this range, it is easier to manufacture by the manufacturing method described later, and the Sdq and Sdr of the gas permeable film tend to fall within the desired range.

[0017] On the other hand, the components other than the 4-methyl-1-pentene polymer that the gas permeable film may contain are not particularly limited and can be known additives, etc.

[0018] In this embodiment, the gas permeable film has a root mean square slope (Sdq) of at least one surface that is 0.0000 or more and 0.0100 or less, and a developed area ratio (Sdr) that is 0.0000 or more and 0.0070 or less. In this embodiment, it is sufficient if at least one surface of the gas permeable film satisfies the above Sdq and Sdr, but it is more preferable if both surfaces of the gas permeable film satisfy the above Sdq and Sdr. When the gas permeable film is used in a culture vessel, it is preferable to observe or optically detect cells cultured in the culture vessel within the culture vessel. In this case, the gas permeable film of this embodiment is usually adjacent to cells in the culture vessel. Therefore, if the gas permeable film has irregularities, these irregularities will be picked up during observation or optical detection, making it difficult to perform the above observation or detection accurately. In contrast, if the Sdq and Sdr of the gas permeable film are within the above range, it becomes possible to reliably observe and detect cells.

[0019] Here, the Sdq of at least one surface of the gas permeable film should be between 0.0000 and 0.0100, preferably between 0.0000 and 0.0030, and more preferably between 0.0001 and 0.0050. Sdq is a value measured in accordance with ISO 25178-2:2012. This Sdq is a value that can be determined, for example, by acquiring the surface state of a 600 μm × 600 μm area using a three-dimensional optical interferometry system (VS1800 manufactured by Hitachi High-Tech Corporation) and analyzing it. In this embodiment, three Sdq values ​​are identified on one surface of the gas permeable film, and the average of these values ​​is taken as the Sdq of that surface. This Sdq can be adjusted by methods such as the manufacturing method of the gas permeable film described later.

[0020] On the other hand, the Sdr of at least one surface of the gas permeable film may be 0.0000 or more and 0.0070 or less, preferably 0.0000 or more and 0.0030 or less, and more preferably 0.0001 or more and 0.0010 or less. Sdr can be measured in accordance with ISO 25178-2:2012. This Sdr is a value that can be determined by acquiring the surface state of a 600 μm × 600 μm area using, for example, a three-dimensional optical interferometry system (VS1800 manufactured by Hitachi High-Tech Corporation) and analyzing it. In this embodiment, three Sdr values ​​are identified on one surface of the gas permeable film, and the average value of these values ​​is taken as the Sdr of that surface.

[0021] Furthermore, the oxygen permeability of the above gas-permeable film at 23°C, measured in accordance with JIS K7126-1:2006, is 1.0 L / (m³). ２ ×24h×atm) or more 100L / (m ２ It is sufficient if it is less than or equal to (x 24h x atm), and 5.0 L / (m ２ ×24h×atm) or more 100.0L / (m ２ Preferably less than 24 hours atm, and 10.0 L / (m³) ２ ×24h×atm) or more 100.0L / (m ２ A value of less than or equal to 24h × atm is more preferable. When the oxygen permeability is within this range, the gas permeable film can be used for a variety of applications.

[0022] Incidentally, the oxygen permeability is a value measured at a temperature of 23°C and a relative humidity of 0% by the differential pressure type gas permeability measurement method. The equipment used for the measurement is not particularly limited as long as it uses the differential pressure type gas permeability measurement method. For example, BT-3 manufactured by Toyo Seiki Seisakusho can be used. The measurement sample is prepared by cutting out a 90×90 mm test piece from the gas permeable film. Also, the measurement part diameter is 70 mm (the permeation area is 38.46 cm ２ ). When it is predicted that the oxygen permeability is large, it is more preferable to apply an aluminum mask to the measurement sample in advance so that the actual permeation area is 5.0 cm ２ .

[0023] Here, the thickness of the gas permeable film is appropriately selected according to its use, but usually 10 μm or more and 500 μm or less is preferable, and 30 μm or more and 300 μm or less is more preferable. The thicker the gas permeable film, the higher the strength of the gas permeable film, and deformation such as bending can be suppressed. On the other hand, the smaller the thickness of the gas permeable film, the higher the oxygen permeability of the gas permeable film can be increased.

[0024] Also, the haze measured in accordance with JIS K7136:2000 of the gas permeable film is preferably 0.01% or more and 3.00% or less, and more preferably 0.01% or more and 0.10% or less. When the haze of the gas permeable film is within this range, observation of cells and the like in the culture container using the gas permeable film becomes easier. The haze can be measured with a known haze meter (for example, HM-150 manufactured by Murakami Color Technology Laboratory). In this embodiment, the haze at any five locations of the gas permeable film is specified, and the average value thereof is adopted.

[0025] The standard deviation of the brightness of the gas permeable film is preferably between 0.0 and 4.0, and more preferably between 0.0 and 2.0. A smaller standard deviation of brightness of the gas permeable film makes it easier to observe and optically detect cells and other elements in culture vessels using the gas permeable film more accurately. The standard deviation of brightness can be obtained by imaging the permeable film under the following conditions and analyzing the resulting image with image analysis software (ImageJ). (Imaging conditions) Microscope: BZ-X710 (Keyence Corporation) Objective lens: S Plan Fluor ELWD 20X (Nikon Corporation) Exposure time: 1 / 100 s

[0026] The Rockwell hardness of the gas permeable film is preferably between 80 and 115, and more preferably between 90 and 115. When the Rockwell hardness is within this range, the gas permeable film is less likely to be scratched during manufacturing, storage, or processing. Therefore, there is an advantage in that the ability to observe cells under a microscope in a culture vessel using the gas permeable film is not easily impaired. The Rockwell hardness is a value measured on the R scale in accordance with ISO 2039-2:1987.

[0027] The total light transmittance of the gas permeable film, as measured in accordance with JIS K 7361-1:1997, is preferably 70% to 100%, and more preferably 90% to 100%. When the total light transmittance of the gas permeable film is 70% or higher, the observation of cells and other organisms in a culture vessel using the gas permeable film is improved. For example, cells and other organisms can be observed by transmitting light through the gas permeable film, or by observing them through the gas permeable film, further improving the observation and detection of cells and other organisms.

[0028] Furthermore, a smaller in-plane optical phase difference of a gas-permeable film is preferable because it indicates superior optical homogeneity, making it easier to observe cells through the gas-permeable film using a phase-contrast microscope. For example, the in-plane optical phase difference of a gas-permeable film measured at 23°C and a light wavelength of 543 nm is preferably 0 nm to 100 nm, more preferably 0 nm to 50 nm, and even more preferably 0 nm to 10 nm. An example of a device capable of measuring such an in-plane optical phase difference of a gas-permeable film is the WPA-200 two-dimensional birefringence evaluation system manufactured by Photonics Latis.

[0029] 2. Method for Manufacturing Gas Permeable Films The method for manufacturing the gas permeable film described above is not particularly limited, but it can be manufactured by, for example, the following two methods.

[0030] (1) First Method The first method is a method comprising the steps of: preparing a film molding resin composition containing a 4-methyl-1-pentene polymer (preparation step); and forming the molten film molding resin composition into a film by passing it between two rolls (forming step). In this method, the Sdq of the surface of at least one of the two rolls used in the forming step is set to 0.0000 or more and 0.0030 or less, and the Sdr is set to 0.0000 or more and 0.0007 or less. This makes it possible to reflect the shape of the roll surface (Sdq and Sdr) on at least one surface of the gas permeable film.

[0031] In the preparation step, a resin composition for film molding containing a 4-methyl-1-pentene polymer is prepared. This resin composition for film molding may consist only of the above-mentioned 4-methyl-1-pentene polymer, or it may be a mixture containing the above-mentioned 4-methyl-1-pentene polymer and other components. If the resin composition for film molding is a mixture, it may be a dry blend of multiple materials or a melt blend (melt kneading).

[0032] In the forming process, the above-described resin composition for film formation is melted and passed between two opposing rolls. At this time, the temperature of the resin composition for film formation is not particularly limited, and usually, it may be a temperature not lower than the melting point of the 4-methyl-1-pentene polymer. For example, a temperature of 240°C or higher and 300°C or lower is preferable, and a temperature of 240°C or higher and 270°C or lower is more preferable.

[0033] In addition, the material of the roll for forming the resin composition for film formation into a film is not particularly limited, but being made of metal is preferable from the viewpoint of being easy to process Sdq and Sdr into desired values. Further, it is sufficient that Sdq of the surface of at least one of the two opposing rolls is 0.0000 or more and 0.0030 or less, and Sdr is 0.0000 or more and 0.0007 or less. However, from the viewpoint that Sdq and Sdr of both surfaces of the obtained gas permeable film will be within the desired range, it is preferable that Sdq and Sdr of both rolls are within the above range. Regarding Sdq of the above roll, 0.0000 or more and 0.0025 or less is more preferable, 0.0000 or more and 0.0020 or less is more preferable, and 0.0001 or more and 0.0020 or less is even more preferable. Also, regarding Sdr of the above roll, 0.0000 or more and 0.0005 or less is more preferable, 0.0000 or more and 0.0003 or less is more preferable, and 0.0001 or more and 0.0003 or less is even more preferable. Sdr and Sdq are measured by the method described above.

[0034] The apparatus used in the above molding process is not particularly limited, as long as it can process the film-forming resin composition between two rolls having the desired Sdq and Sdr, and process it into a film with the desired thickness and a highly smooth surface (having the desired Sdq and Sdr). For example, a known T-die extrusion molding machine (film / sheet extrusion) can be used. If the apparatus is a T-die extrusion molding machine, the molten film-forming resin composition is discharged from the lip of the T-die. The film-forming resin composition is then pressed against the rolls using a known pressing mechanism (air knife method, vacuum chamber method, electrostatic pinning method, roll pressing method, sleeve touch molding method, etc.). Here, from the viewpoint of pressing the pressing surface of the roll, which has mirror-like (smooth) properties, against the molten film-forming composition using the pressing mechanism, and transferring the shape well, it is desirable that the pressing mechanism be a metal elastic roll pressing method or a sleeve touch molding method that has a surface pressing action.

[0035] Alternatively, instead of using two metal rolls having the surface conditions described above, a separator film made of a smooth, heat-resistant resin having the surface shape (in the range of Sdq or Sdr) described above may be used. In this case, the separator film is placed against the surface of one or both of the two rolls of the apparatus. Then, the separator film and the resin composition for film molding are pressed together to transfer the surface shape of the separator film to the resin composition for film molding. After that, the film with the transferred surface shape (gas-permeable film) is taken up and transported, and the separator film may be peeled off. Preferred resin separator films with such high smoothness and heat resistance include biaxially oriented polyethylene terephthalate film, biaxially oriented polyethylene naphthalate film, and polyimide film obtained by solution casting. The thickness of the separator film is preferably in the range of 10 μm to 100 μm from the viewpoint of strength and flexibility.

[0036] Incidentally, the apparatus may further have two or more rolls as means for taking up the gas permeable film. In this case, by sequentially cooling the resin composition for film forming between the two rolls, it is possible to suppress sticking to the rolls and the like, and it is preferable to obtain a gas permeable film with stable shape dimensions and appearance.

[0037] Further, the molding apparatus may further include means for taking up the gas permeable film after production.

[0038] (2) Second method The second method includes a step of preparing a molded film containing a 4-methyl-1-pentene polymer (film preparation step) and a step of sandwiching the molded film between two plate-like members and pressing it (pressing step). In the pressing step of this method, Sdq of the surface of at least one of the plate-like members facing the film is set to 0.0000 or more and 0.0030 or less, and Sdr is set to 0.0000 or more and 0.0007 or less. Thereby, the surface shape (Sdq and Sdr) of the plate-like member can be reflected on at least one surface of the gas permeable film.

[0039] In the film preparation step, a molded film containing a 4-methyl-1-pentene polymer may be prepared. In the film preparation step, for example, the resin composition for film forming in the above-described first method may be prepared and molded by a known method. The molding method is not particularly limited, and examples thereof include extrusion molding, T-die extrusion molding, injection molding, film molding, inflation molding, blow molding, extrusion blow molding, press molding, calendar roll molding, vacuum molding, pressure air molding, and the like. Further, in the film preparation step, a commercially available molded film containing a 4-methyl-1-pentene polymer may be prepared.

[0040] In the pressing process, the molded film is sandwiched between two plate-like members and pressed. The material of the plate-like members is not particularly limited, but it is preferable that they be made of metal, as this makes it easier to process Sdq and Sdr to desired values. Furthermore, it is sufficient that the Sdq of the surface of at least one of the two plate-like members facing the molded film is 0.0000 or more and 0.0030 or less, and the Sdr is 0.0000 or more and 0.0007 or less. However, it is preferable that the Sdq and Sdr of the surfaces of both plate-like members (the surfaces facing the molded film) are within the above range, as this ensures that the Sdq and Sdr of both sides of the resulting gas permeable film are within the desired range. The Sdq of the surface of the plate-like members is more preferably 0.0000 or more and 0.0025 or less, more preferably 0.0000 or more and 0.0020 or less, and more preferably 0.0001 or more and 0.0020 or less. Furthermore, the Sdr of the plate-like member is more preferably 0.0000 to 0.0005, more preferably 0.0000 to 0.0003, and more preferably 0.0001 to 0.0003. Sdr and Sdq are measured by the method described above.

[0041] During the heating press process, the temperature of the plate-shaped member is preferably 240°C to 300°C, and more preferably 240°C to 270°C. When the temperature of the plate-shaped member is within this range, the surface shape of the plate-shaped member is more easily transferred to the molded film. Furthermore, the pressure during pressing is preferably 0.1 MPa to 3.0 MPa, and more preferably 0.1 MPa to 1.0 MPa. In addition, the pressing time is preferably 60 seconds to 200 seconds, and more preferably 120 seconds to 200 seconds. When the pressure and time are within this range, the surface shape of the plate-shaped member is more easily transferred to the molded film. Next, the plate-shaped member is cooled to room temperature in the cooling press process.

[0042] The equipment used in the above pressing process can be a known press or the like. The equipment may further include means for transporting the molded film and means for winding up the gas-permeable film after manufacturing (after pressing).

[0043] 3. Culture Vessels The present invention also provides culture vessels for static culture of cells, microorganisms, etc. In this specification, the culture of cells and microorganisms means to increase, grow, or maintain them in a viable state. Static culture means culturing microorganisms without applying artificial shaking, such as with a microplate shaker.

[0044] The culture vessel only needs to include the gas permeable film described above. Figure 1 is a perspective view of the culture vessel 100, and Figure 2 is a cross-sectional view of a part of the culture vessel 100. However, the culture vessel 100 is just one example of a culture vessel in this embodiment and is not limited to this structure.

[0045] The culture vessel 100 shown in Figure 1 has, for example, a plurality of containment sections 210 (see Figure 2) for culturing cells or the like. The culture vessel 100 is placed in the culture space of a culture device (for example, a constant temperature chamber) with the liquid culture medium and the cells or the like to be cultured (hereinafter also referred to as "subjects") contained in the containment sections 210. Note that the culture vessel 100 does not need to be used while placed in the culture device. The culture vessel 100 may be used in various situations depending on the subject.

[0046] The culture vessel 100 has a main body portion 220 having a plurality of through holes 224, and a resin sheet 230 made of the aforementioned gas permeable film that closes one end of the through holes 224.

[0047] The main body 220 is made of, for example, a thermoplastic resin and is an integrally molded product produced by injection molding. Examples of thermoplastic resins that make up the main body 220 include polystyrene and polyolefins. Examples of polyolefins include polyethylene, polypropylene, 4-methyl-1-pentene (co)polymers, and cyclic olefin (co)polymers. Of these, 4-methyl-1-pentene polymers are preferred from the viewpoint of improving the gas permeability of the main body 220. Alternatively, from the viewpoint of improving the heat resistance of the main body 220, 4-methyl-1-pentene polymers, polystyrene, polypropylene, and cyclic olefin polymers are preferred.

[0048] The resin sheet (the gas-permeable film described above) 230 is attached to the main body 220 via an adhesive layer 240 so as to cover (close) the through holes 224. In this embodiment, the resin sheet 230 closes the bottom surface of the main body 220 and constitutes the bottom surface of the containment section 210 (culture container 100). In other words, the area covered by the walls of the through holes 224 of the main body 220 and the resin sheet 230 becomes the containment section 210. In this embodiment, since the resin sheet 230 is made of the gas-permeable film described above, the resin sheet 230 also functions as an oxygen-permeable layer that supplies oxygen to the object being cultured inside the containment section 210. In this embodiment, a single resin sheet 230 closes multiple through holes 224 of the main body 220, but different resin sheets 230 may be placed for each individual through hole 224.

[0049] The adhesive layer 240 adheres to the lower surface of the bottom plate portion of the housing portion 210 by attaching the resin sheet 230. Known adhesives such as acrylic, silicone, urethane, and rubber-based adhesives can be used for the adhesive layer 240. Of these, acrylic and silicone-based adhesives are preferred.

[0050] Each well 210 in this embodiment is cylindrical and opens upward. In this embodiment, the plan view shape of the opening of the well 210 is circular. However, the structure is not limited to this. For example, a resin sheet (for example, the gas permeable film described above) may also be placed on the top surface. In addition, although the wells 210 are arranged in the left-right and front-back directions, only one well may be provided. The number of wells 210 in the culture container 100 is preferably 5 or more, more preferably 80 or more, and even more preferably 300 or more. For example, the number of wells 210 may be 6, 24, 96, or 384, or any other number.

[0051] Furthermore, the culture container 100 may have a coating layer 300 made of a hydrophilic material on the inner surface of the containment section 210 to enhance the adhesion of the object to it.

[0052] 4. Observation Method Using the Culture Vessel Described The culture vessel 100 described above uses the resin sheet (gas permeable film) 230 with minimal irregularities described above as a component for the bottom surface of the containment section 210. Therefore, when observing cells, microorganisms, or other target substances in the containment section 210 of the culture vessel 100 without retrieving the substances from the containment section 210, the irregularities of the resin sheet 230 adjacent to the cells are difficult to observe. Therefore, observation and measurement of cells and other substances can be performed more accurately. Note that observation and measurement of cells and other substances may be performed by placing a microscope or measuring device on the bottom surface and using the resin sheet 230 as a backdrop. Alternatively, a microscope or measuring device may be placed on the top surface and observation or optical measurement may be performed with the resin sheet 230 as a background. Furthermore, a reflective material for reflecting light may be placed on the bottom or top surface, and observation or optical measurement may be performed using reflected light.

[0053] There are no particular restrictions on the types of observation or optical measurement methods, and observation using a microscope is acceptable. The devices used for measurement methods can be known microscopes such as biological microscopes, optical phase-contrast microscopes, differential interference microscopes, and confocal microscopes, and there are no particular restrictions on differences in imaging, structural, or illumination methods. For example, imaging methods can be optical, electronic, or scanning probe; structural imaging can be upright, inverted, or stereo; and illumination can be transmitted or reflected. Furthermore, a device integrating a camera and image analysis system is also acceptable.

[0054] 5. Biomimetic Systems The culture vessels described above can also be used in biomimetic systems (Micro-Physiological Systems: MPS). A biomimetic system is a cell culture system that reproduces tissue functions in vivo by arranging organs and tissue compartments that mimic the physiological environment of various organs such as the liver, small intestine, lungs, and heart, either individually or in combination.

[0055] The biomimetic system may include a culture vessel having at least a portion of the aforementioned gas-permeable film. For example, it may have a structure in which multiple culture vessels are combined.

[0056] Examples of such biomimetic systems include co-culture systems combining a plate with the aforementioned gas-permeable film on its bottom surface with a cell culture insert container, and fluid channel devices with fluid channels arranged within the culture vessel. However, the system is not limited to these structures and can be used in biomimetic systems of various structures.

[0057] The gas-permeable film described above has high gas permeability and a smooth surface. Therefore, a biomimetic system including this gas-permeable film (culture vessel) can be used to culture various organs and tissue compartments. Furthermore, the smooth surface of the gas-permeable film has the advantage of making it easy to observe the organs and tissue compartments cultured on it. In addition, because the gas-permeable film has excellent surface smoothness, it has low sorption of drugs used, for example, in drug discovery screening. Therefore, it can be used to create a biomimetic system with an environment more suitable for culturing organs and tissue compartments.

[0058] The present invention will be described below with reference to examples. The scope of the present invention is not to be limited by the examples.

[0059] (1) Preparation of materials The following two types of 4-methyl-1-pentene polymers were prepared: (a-1): Copolymer of 4-methyl-1-pentene and 1-decene (amount of constituent units derived from 4-methyl-1-pentene: 98.4 mol%, amount of constituent units derived from 1-decene: 1.6 mol%), MFR: 180 g / 10 min (260°C, 5.0 kg load) (a-2): Copolymer of 4-methyl-1-pentene and 1-decene (amount of constituent units derived from 4-methyl-1-pentene: 97.9 mol%, amount of constituent units derived from 1-decene: 2.1 mol%) MFR: 25 g / 10 min (260°C, 5.0 kg load) The amounts of each constituent unit in the above 4-methyl-1-pentene polymers are as follows: １３ The quantification was performed by 13C-NMR. The melt flow rate (MFR) was measured at 260°C and a 5.0 kg load, in accordance with ASTM D1238.

[0060] (2) Manufacturing of gas permeable film (Example 1) A single-screw extruder (cylinder inner diameter D: 20 mm, full-flight screw, L / D: 28 mm when the effective screw length is L) was used as the molding machine, and the apparatus was equipped with a T-die (die width: 300 mm, lip opening: 0.4 to 1.0 mm), a cooling roll (outer diameter 200 mm, made of steel with mirror-finish hard chrome plating surface treatment, water-cooled), and a take-up machine. The above-mentioned 4-methyl-1-pentene polymer (a-1) was put into the hopper of the apparatus. The temperature of each part of the cylinder of the apparatus was set to 240 to 250°C, the screw rotation speed to 40 to 70 rpm, and the 4-methyl-1-pentene polymer (a-1) was melted and kneaded. Then, it was extruded from the T-die at an extrusion rate of 1.1 to 1.5 kg / hour.

[0061] The extruded film was cooled using a cooling roll (with internal water temperature at 20°C), and then taken up using a take-up machine (take-up speed: 0.4-0.6 m / min) to obtain a molded film with a sheet width of approximately 270 mm and a thickness of 50 μm.

[0062] A polyimide resin film (manufactured by UBE, brand name: Upirex-S, (root mean square slope (Sdq): 0.0020, unfolded area ratio (Sdr): 0.0002)) and an aluminum plate-shaped member were placed on both sides of the obtained molded film, starting from the surface in contact with the film. The mixture was heated and pressurized at 240°C for 180 seconds at a pressure of 0.1 MPa, and then cooled to room temperature using a cooling press. This yielded the gas-permeable film of Example 1 with a thickness of 50 μm.

[0063] (Example 2) As a molding machine, a device was used that included a single-screw extruder (cylinder inner diameter D: 50 mm, full-flight screw, L / D: 32 mm when the effective screw length is L, with a gear pump), a T-die (die width: 600 mm, lip opening: 0.5 mm), a sleeve touch roll (sleeve thickness: 300 μm, two support rolls with a diameter of 200 mm, root mean square slope (Sdq) of the surface: 0.0020, developed area ratio (Sdr): 0.0002), a cooling roll (outer diameter 300 mm, made of steel with a mirror-finish hard chrome plated surface treatment, water-cooled), and a take-up machine. The above-mentioned 4-methyl-1-pentene polymer (a-1) was put into the hopper of the device. The temperature of each part of the cylinder of the device was set to 260 to 295°C, the screw rotation speed to 30 to 60 rpm, and the 4-methyl-1-pentene polymer (a-1) was melted and kneaded. Then, the material was extruded from the T-die at an extrusion rate of 6.0 to 9.0 kg / hour.

[0064] (Example 3) As a molding machine, a device was used that included a single-screw extruder (cylinder inner diameter D: 50 mm, full-flight screw, L / D: 32 mm when the effective screw length is L, with a gear pump), a T-die (die width: 600 mm, lip opening: 0.5 mm), a sleeve touch roll (sleeve thickness: 300 μm, two support rolls with a diameter of 200 mm, root mean square slope (Sdq) of the surface: 0.0020, developed area ratio (Sdr): 0.0002), a cooling roll (outer diameter 300 mm, made of steel with a mirror-finish hard chrome plated surface treatment, water-cooled), and a take-up machine. The above-mentioned 4-methyl-1-pentene polymer (a-1) was put into the hopper of the device. The temperature of each part of the cylinder of the device was set to 260 to 290 °C, the screw rotation speed to 80 to 110 rpm, and the 4-methyl-1-pentene polymer (a-1) was melted and kneaded. Then, the material was extruded from the T-die at an extrusion rate of 15.5 to 17.5 kg / hour.

[0065] (Comparative Example 1) A gas permeable film with a thickness of 50 μm was prepared in the same manner as in Example 1, except that heating and cooling pressing were not performed on both sides of the molded film.

[0066] (Comparative Example 2) A gas permeable film with a thickness of 50 μm was prepared in the same manner as in Comparative Example 1, except that a 4-methyl-1-pentene polymer (a-2) was used as the 4-methyl-1-pentene polymer.

[0067] (3) Evaluation and Surface Condition (Sdq and Sdr) For the gas permeable films prepared in the examples and comparative examples, the surface condition was acquired for a 600 μm × 600 μm area using a three-dimensional optical interferometry system (VS1800, Hitachi High-Tech Corporation) in accordance with ISO 25178-2:2012. From the obtained surface condition, the root mean square slope (Sdq) and the unfolded area ratio (Sdr) were determined. For each of the three locations on one gas permeable film, the root mean square slope (Sdq) and the unfolded area ratio (Sdr) were determined, and their average values ​​were adopted as Sdq and Sdr, respectively. The observation conditions for the three-dimensional optical interferometry system were set as follows: (Observation conditions) Objective lens: x10 Intermediate lens: x1 Measurement mode: Wave Surface correction: 4th order Number of measurement points: n=1

[0068] - The gas permeable films prepared in the oxygen permeability examples and comparative examples were cut to a size of 90 mm wide x 90 mm long to serve as test specimens. A circular measurement section of the test specimen with a diameter of 70 mm (permeable area: 38.46 cm²) was used. ２ Regarding ), the oxygen permeability [L / (m³)] was measured in accordance with JIS K7126-1:2006 using a differential pressure type gas permeability measuring device (Toyo Seiki Seisakusho Co., Ltd., BT-3) at a temperature of 23°C and humidity of 0%. ２ The following was measured: (24h, atm).

[0069] For the gas-permeable films prepared in the haze examples and comparative examples, the haze was measured at five arbitrary locations using a haze meter (HM-150, manufactured by Murakami Color Technology Research Institute Co., Ltd.) in accordance with JIS K7136:2000. The average value of these measurements was then adopted as the haze (%).

[0070] For the gas permeable films prepared in the Rockwell hardness examples and comparative examples, measurements were performed on a 100 mm (width) x 100 mm (length) x 10 mm (thickness) resin test piece using the R scale, in accordance with ISO 2039-2:1987.

[0071] - Ease of observation during cell culture (brightness) <Preparation of cell culture vessels> The gas permeable films prepared in the examples and comparative examples were cut to a size of 8 cm x 12 cm, and attached to the bottom of a 24-well polystyrene container frame (main body) via a thin double-sided adhesive (3M, acrylic, brand name: 93005LE) to create culture plates (culture vessels). The culture vessels were then packed in gamma-ray resistant bags and sterilized by irradiation with 15 kGy of gamma rays. A 0.001 M hydrochloric acid solution was prepared by diluting a 0.1 M hydrochloric acid solution (for volumetric analysis, Fujifilm Wako Pure Chemical Industries, Ltd.) 100-fold with sterile water for injection (Japanese Pharmacopoeia, Otsuka Pharmaceutical Co., Ltd.) to prepare a 0.001 M hydrochloric acid solution for each well of the sterilized cell culture vessel. The prepared hydrochloric acid solution was sterilized by filtration. A 3 mg / mL collagen solution (Cell Matrix Type I-P, manufactured by Nitta Gelatin Co., Ltd., derived from pig tendons) was diluted with the above 0.001 M hydrochloric acid solution to obtain a coating material. Each coating material was then applied using a pipette to a volume of 200 μl. The containers were then dried overnight at room temperature to obtain containers with the coated layers.

[0072] <Cell Culture> Cell culture was carried out in the wells of each culture vessel under the following conditions: (Culture conditions) Cells: TFK-1 cells Number of cells: 1 × 10 ４ cell / cm ２ Culture medium volume: 0.5 mL / well; Culture environment temperature: 37°C; CO2 level of the culture environment ２ Concentration: 5% Humidity of culture environment: 100% Culture period: 3 days

[0073] <Imaging> After the culture process described above, observation and imaging were performed from the gas-permeable film side under the following conditions. (Imaging conditions) Microscope: BZ-X710 (Keyence Corporation) Objective lens: S Plan Fluor ELWD 20X (Nikon Corporation) Exposure time of culture vessel: 1 / 100 s

[0074] <Image Analysis> Of the images obtained above, the portion without cells was selected for analysis, and the standard deviation of the brightness of the gas permeable film was analyzed using image analysis software (ImageJ) under the following conditions. (Analysis Conditions) The image to be analyzed (hereinafter also referred to as the "analysis target image") was opened in the analysis software. Next, ImageJ was used to select the portion to be analyzed from the analysis target image (hereinafter also referred to as the "analysis target portion"). Next, ImageJ was used to calculate the mean value and standard deviation (StdDev) of the gray value (brightness) of the analysis target portion.

[0075] - Cell culture and observation by co-culture <Preparation of cell culture vessels> The gas-permeable films prepared in Example 1 were each cut to a size of 8 cm x 12 cm and attached to the bottom of a 24-well polystyrene container frame (main body) using a thin double-sided adhesive (3M, acrylic, brand name: 93005LE) to create culture plates (culture vessels). The culture vessels were then packed in gamma-ray resistant bags and sterilized by irradiation with 15 kGy of gamma rays. A 0.001 M hydrochloric acid solution was prepared by diluting a 0.1 M hydrochloric acid solution (for volumetric analysis, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) 100-fold with sterile water for injection (Japanese Pharmacopoeia, manufactured by Otsuka Pharmaceutical Co., Ltd.) to prepare a 0.001 M hydrochloric acid solution for each well of the sterilized cell culture vessel. The prepared hydrochloric acid solution was sterilized by filtration. A 3 mg / mL collagen solution (Cell Matrix Type I-P, manufactured by Nitta Gelatin Co., Ltd., derived from pig tendons) was diluted with the above 0.001 M hydrochloric acid solution to obtain a coating material. Each coating material was then applied using a pipette to a volume of 200 μl. The containers were then dried overnight at room temperature to obtain containers with the coated layers.

[0076] <Preparation of comparative cell vessels> Cell culture vessels were prepared in the same manner as above, except that a film made of polystyrene was used instead of the gas-permeable film prepared in Example 1.

[0077] <Insert container> Falcon® Culture Insert, 24-well, 0.4 μm PET transparent membrane, product number 353095 was used.

[0078] <Cell Culture> Cell culture was performed in the wells of each culture vessel under the following conditions: (Insert culture conditions) Cells: A549 cells Cell count: 1.27 × 10 ５ cell / cm ２ Culture medium volume: 0.3 mL / well; Culture environment temperature: 37°C; CO2 level of the culture environment ２ Concentration: 5% Humidity of culture environment: 100% Culture period: 7 days

[0079] (Insert culture conditions) Cells: HepG2 cells Cell count: 2.00 × 10 ４ cell / cm ２ Culture medium volume: 0.5 mL / well; Culture environment temperature: 37°C; CO2 level of the culture environment ２ Concentration: 5% Humidity of culture environment: 100% Culture period: 2 days (5 days of culture on insert, then cell seeding)

[0080] <Imaging> After the culture was performed as described above, observation was carried out from the gas-permeable film side under the following conditions, and imaging was performed under the following conditions. Figure 3A shows a photograph under the insert when co-culturing was performed using the gas-permeable film of Example 1. Figure 3B shows a photograph under the insert when co-culturing was performed using polystyrene film. (Imaging conditions) Microscope: Olympus CKX43 Objective lens: 10X (Field of view: 22)

[0081] <Evaluation> The cell culture performance and imaging results described above were evaluated as follows. The results when using the gas permeable film of Example 1 are shown in Table 1. Although not shown in the table, the evaluation when using polystyrene film was △. ○: Cells above and below the insert adhered and spread sufficiently, and this was uniform within and between wells, and the cells were clearly observed. △: Cells above and below the insert adhered and spread, and this was uniform within and between wells, and the cells were clearly observed. ×: Cells did not adhere and formed cell clumps in most areas of the entire well above and below the insert.

[0082] (4) Results

[0083] As shown in Table 1 above, when a gas permeable film with a root mean square slope (Sdq) of 0.0000 or more and a developed area ratio (Sdr) of 0.0000 or more and 0.0070 or less is used in a cell culture vessel, brightness unevenness is less likely to occur (Examples 1-3), and cell observation through the gas permeable film is easier. Furthermore, the evaluation results for cell culture and observation during co-culture were good. In addition, as shown in Figure 3A, the adhesion and proliferation of HepG2 cells in the insert-under-well plate were significantly better compared to when the styrene film shown in Figure 3B was used (Example 1).

[0084] On the other hand, when the root mean square slope (Sdq) or the area ratio (Sdr) did not meet the above range, the standard deviation of brightness was large (Comparative Examples 1 and 2), making it difficult to observe cells through the gas-permeable film.

[0085] This application claims priority under Japanese Patent Application No. 2025-014140, filed on 30 January 2025. All contents described in the specification and drawings of said application are incorporated herein by reference.

[0086] The gas-permeable film of the present invention is a gas-permeable film that can be used in cell culture vessels, and allows observation of cells and other elements through the film. Therefore, it is extremely useful in fields such as medicine and pharmacy.

[0087] 100 Culture container 210 Storage section 220 Main body 224 Through hole 230 Resin sheet 240 Adhesive layer 300 Coating layer

Claims

1. A gas permeable film containing a 4-methyl-1-pentene polymer, wherein the root mean square slope (Sdq) of at least one surface, measured in accordance with ISO 25178-2:2012, is 0.0000 or more and 0.0100 or less, and the unfolded area ratio (Sdr), measured in accordance with ISO 25178-2:2012, is 0.0000 or more and 0.0070 or less, and the oxygen permeability at a temperature of 23°C and humidity of 0%, measured in accordance with JIS K7126-1:2006, is 1.0 L / (m²). ２ ・24h・atm) or more 100.0L / (m ２ A gas-permeable film with a humidity level of 24h·atm or less.

2. The gas permeable film according to claim 1, wherein the 4-methyl-1-pentene polymer comprises a structural unit derived from 4-methyl-1-pentene and a structural unit derived from ethylene and / or an α-olefin having 3 to 20 carbon atoms (excluding 4-methyl-1-pentene).

3. The gas permeable film according to claim 1, wherein when the total constituent units of the 4-methyl-1-pentene polymer are set to 100 mol%, the amount of constituent units derived from 4-methyl-1-pentene is 60 mol% or more and 99 mol% or less.

4. The gas permeable film according to claim 1, wherein the thickness is 10 μm or more and 500 μm or less.

5. The gas permeable film according to claim 1, wherein the standard deviation of luminance is 0.0 or more and 4.0 or less.

6. The gas permeable film according to claim 1, wherein the haze measured in accordance with JIS K7136:2000 is 0.01% or more and 3.00% or less.

7. The gas permeable film according to claim 1, wherein the Rockwell hardness measured on the R scale in accordance with ISO 2039-2:1987 is 80 or more and 115 or less.

8. A method for producing a gas permeable film, comprising the steps of: preparing a resin composition for film molding containing a 4-methyl-1-pentene polymer; and forming the molten resin composition for film molding into a film by passing it between two opposing rolls, wherein the root mean square slope (Sdq) of the surface of at least one of the two opposing rolls, measured in accordance with ISO 25178-2:2012, is 0.0000 or more and 0.0030 or less, and the developed area ratio (Sdr), measured in accordance with ISO 25178-2:2012, is 0.0000 or more and 0.0007 or less.

9. The method for producing a gas permeable film according to claim 8, wherein the melt flow rate of the 4-methyl-1-pentene polymer at 260°C under a 5.0 kg load is 5 g / 10 min or more and 180 g / 10 min or less.

10. A method for manufacturing a gas permeable film, comprising the steps of: preparing a film containing a 4-methyl-1-pentene polymer; and sandwiching the film between two plate-like members and pressing it, wherein the root mean square slope (Sdq) of at least one of the two plate-like members facing the film, as measured in accordance with ISO 25178-2:2012, is 0.0000 or more and 0.0030 or less, and the developed area ratio (Sdr), as measured in accordance with ISO 25178-2:2012, is 0.0000 or more and 0.0007 or less.

11. A culture vessel using a gas-permeable film according to any one of claims 1 to 7.

12. The culture vessel according to claim 11, comprising: a main body having a through hole; and the gas permeable film attached to the main body so as to close one end of the through hole.

13. A biomimetic system comprising the culture vessel of claim 11.

14. An observation method comprising the steps of: culturing cells or microorganisms in a containment section surrounded by the wall surface of the through-hole and the gas permeable film of the culture vessel according to claim 12; and observing the cells or microorganisms.

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