Multilayer film, container and method for manufacturing the same, and method for culturing cells

Abstract

To provide a multilayer film that has high oxygen permeability and can be heat-sealed to form a container that is resistant to tearing from impacts such as dropping when it contains liquid. [Solution] A heat seal layer comprising copolymer A, which is a copolymer of 4-methyl-1-pentene and propylene, further comprising copolymer B, which is a copolymer of ethylene and an α-olefin having 3 to 20 carbon atoms, or copolymer C, which is a copolymer of propylene and an α-olefin (excluding propylene) having 2 to 20 carbon atoms; and a core layer comprising (co)polymer D, which is a 4-methyl-1-pentene (co)polymer, wherein the film impact strength measured from the opposite side of the heat seal layer in accordance with ASTM-D 3420:2021 is 7 kJ / m or more, and the oxygen permeability at 23°C is 3.0 L / (m 2 ·24h·atm) or more 100.0L / (m 2 Multilayer film with a temperature of 24 hours (atm) or less.

Description

[Technical Field]

[0001] The present invention relates to multilayer films, containers, methods for manufacturing the same, and methods for culturing cells. [Background technology]

[0002] Cell containers are known for propagating, growing, storing, and transporting cells, in which an oxygen-permeable film is heat-sealed to create an airtight container, and a port member attached to a part of the container connects the inside and outside of the container (for example, Patent Document 1). Patent Document 1 states that linear low-density polyethylene (LLDPE) and polypropylene can be suitably used as the oxygen-permeable film. [Prior art documents] [Patent Documents]

[0003] [Patent Document 1] International Publication No. 2022 / 014436 [Overview of the Initiative] [Problems that the invention aims to solve]

[0004] Cell vessels are made of oxygen-permeable materials such as LLDPE as described in Patent Document 1. However, if the oxygen permeability of the cell vessels can be further increased, it is expected that the efficiency of cell culture will also be increased.

[0005] Furthermore, cell containers are used to contain cells along with a liquid such as a culture medium and to culture the cells within the container. According to the inventors' new findings, when a cell container is accidentally dropped during transport or storage while cell culture is in progress, the cell container may rupture due to the impact, causing the culture medium and cells it contained to leak out.

[0006] The present invention has been made in view of the above problems of the prior art, and aims to provide a multilayer film capable of forming, by heat sealing, a container having high oxygen permeability and being difficult to break even by an impact such as dropping when containing a liquid inside, a container formed from the multilayer film, a method for manufacturing the container, and a method for culturing cells using the cell container.

Means for Solving the Problems

[0007] One aspect of the present invention for solving the above problems relates to the multilayer film, container, and method for manufacturing the same, and method for culturing cells described in the following [1] to

[18] . [1] A heat-sealing layer containing copolymer A which is a copolymer of 4-methyl-1-pentene and propylene, and further containing copolymer B which is a copolymer of ethylene and an α-olefin having 3 or more and 20 or less carbon atoms, or copolymer C which is a copolymer of propylene and an α-olefin (excluding propylene) having 2 or more and 20 or less carbon atoms, A core layer containing (co)polymer D which is a 4-methyl-1-pentene (co)polymer, The film impact strength measured from the opposite side of the heat-sealing layer in accordance with ASTM-D 3420:2021 is 7 kJ / m or more, The oxygen permeability at 23°C is 3.0 L / (m 2 ·24h·atm) or more and 100.0 L / (m 2 ·24h·atm) or less, Multilayer film. [2] The Young's modulus at 23°C measured in accordance with JIS K 6781:1994 is 100 MPa or more and 500 MPa or less, The multilayer film according to [1]. [3] The tensile elongation at break at 23°C measured in accordance with JIS K 6781:1994 is 300% or more and 1000% or less, The multilayer film according to [1] or [2]. [4] The haze measured in accordance with ASTM D-1003:2021 is 0% or more and 40% or less, A multilayer film as described in any of [1] to [3]. [5] The thickness is 50 μm or more and 1 mm or less. A multilayer film as described in any of [1] to [4]. [6] The ratio of the thickness of the heat seal layer to the thickness of the core layer (heat seal layer / core layer) is 1 / 10 or more and 5 / 1 or less. A multilayer film as described in any of [1] to [5]. [7] The sterile assurance level (SAL) of the medical device, as measured in accordance with BS EN556-1:2001, is 10 -3 The following is: A multilayer film as described in any of [1] to [6]. [8] The heat seal strength when the heat seal layers are heat-sealed together at 150°C is 10 N / 15 mm or more and 300 N / 15 mm or less. A multilayer film as described in any of [1] to [7]. [9] The heat seal layer has a sea-island structure, A multilayer film as described in any of [1] to [8].

[10] The copolymer A comprises 70 mol% to 99 mol% of 4-methyl-1-pentene and 1 mol% to 30 mol% of propylene relative to the total number of constituent units. A multilayer film as described in any of [1] to [9].

[11] The heat seal layer contains 3% by mass or more and 30% by mass or less of copolymer B and copolymer C with respect to the total mass of copolymer A, copolymer B and copolymer C. A multilayer film as described in any of [1] to

[10] .

[12] The core layer comprises a copolymer of ethylene and an α-olefin having 3 to 20 carbon atoms, or a copolymer of propylene and an α-olefin (excluding propylene) having 2 to 20 carbon atoms. A multilayer film as described in any of [1] to

[11] .

[13] Used in the formation of cell vessels, A multilayer film as described in any of [1] to

[12] .

[14] One or more multilayer films according to any of [1] to

[13] are heat-sealed together to form a bag, container.

[15] Having a port member that connects the inside and outside of the container, The port member is bonded to the multilayer film in the heat-sealed portion of the heat-seal layer. The container described in

[14] .

[16] A cell container, as described in

[14] .

[17] A method for manufacturing a container, comprising the step of heat-sealing one or more multilayer films described in any of [1] to

[13] to form a bag. A step of introducing cells into a cell container containing a multilayer film as described in any of

[18] [1] to

[13] , The process of culturing cells in the aforementioned cell vessel, A method for culturing cells, comprising the following characteristics. [Effects of the Invention]

[0008] The present invention provides a multilayer film that has high oxygen permeability and can be formed by heat sealing to create a container that is resistant to tearing from impacts such as dropping when it contains liquid inside, a container formed from the multilayer film, a method for manufacturing the container, and a method for culturing cells using the cell container. [Brief explanation of the drawing]

[0009] [Figure 1] Figure 1A is a plan view showing the appearance of a cell vessel manufactured from a multilayer film according to one embodiment of the present invention, and Figure 1B is a partial cross-sectional view of the cell vessel shown in Figure 1A along the dashed line 1B-1B. [Modes for carrying out the invention]

[0010] 1. Multilayer film A first embodiment of the present invention relates to a multilayer film. The multilayer film comprises a heat-seal layer and a core layer. The multilayer film may have other layers different from the heat-seal layer and the core layer. For example, the multilayer film may have a skin layer for protecting the surface. In this case, the heat-seal layer may be provided on one surface of the core layer and the skin layer on the other surface. The skin layer may have the same composition and properties as the heat-seal layer.

[0011] 1-1. Heat seal layer A heat-seal layer is a layer that can be fused together by overlapping different parts of the same heat-seal layer, or by overlapping it with another heat-seal layer, and then heating and pressurizing it.

[0012] 1-1-1.Copolymer A~Copolymer C The heat seal layer contains a copolymer of 4-methyl-1-pentene and propylene (hereinafter simply referred to as "Copolymer A"). The heat seal layer further contains a copolymer of ethylene and an α-olefin having 3 to 20 carbon atoms (hereinafter simply referred to as "Copolymer B"), or a copolymer of propylene and an α-olefin (excluding propylene) having 2 to 20 carbon atoms (hereinafter simply referred to as "Copolymer C").

[0013] Copolymer A enhances the oxygen permeability of the heat-seal layer.

[0014] Copolymers B and C make the heat-seal layer relatively flexible, thereby enhancing its stress absorption. This improves the impact resistance of containers molded from multilayer films, particularly those containing copolymers B and C. Therefore, containers molded from these multilayer films are less likely to tear from impacts such as drops, even when filled with liquid.

[0015] According to the inventors' findings, containers molded from multilayer films often have weak impact resistance in the heat-sealed portion of the heat-seal layer (the adhesive portion 116 in Figures 1A and 1B, described later), and are prone to tearing at this heat-sealed portion. Therefore, by making the heat-seal layer more resistant to tearing even when subjected to impact, the impact resistance of the container can be efficiently improved.

[0016] Copolymer A is a copolymer of 4-methyl-1-pentene and propylene.

[0017] Copolymer A may be a binary copolymer having only structural units derived from 4-methyl-1-pentene and structural units derived from propylene, or it may be a ternary copolymer or a more complex copolymer having structural units derived from monomers other than propylene. The monomer other than propylene is preferably an α-olefin having 4 to 20 carbon atoms, different from 4-methyl-1-pentene and propylene. Examples of the α-olefins include ethylene, 1-butene, 1-pentene, 1-hexene, 1-octene, 1-decene, 1-undecene, 1-dodecene, 1-tetradecene, 1-hexadecene, 1-octadecene, and 1-eicosene.

[0018] The proportion of structural units derived from 4-methyl-1-pentene to the total structural units of copolymer A is more preferably 70 mol% to 99 mol%, even more preferably 75 mol% to 99 mol%, and particularly preferably 80 mol% to 99 mol%. The proportion of structural units derived from propylene to the total structural units of copolymer A is preferably 1 mol% to 55 mol%, more preferably 1 mol% to 30 mol%, even more preferably 1 mol% to 25 mol%, and even more preferably 1 mol% to 20 mol%. From the viewpoint of improving the oxygen permeability of the multilayer film, it is preferable that copolymer A has a high proportion of structural units derived from 4-methyl-1-pentene.

[0019] The copolymer B is a copolymer of ethylene and an α-olefin having 3 to 20 carbon atoms. Examples of the α-olefin other than the above ethylene include propylene, 1-butene, 1-pentene, 1-hexene, 3-methyl-1-butene, 3,3-dimethyl-1-butene, 4-methyl-1-pentene, 1-octene, 1-decene, and 1-dodecene. Among these, from the viewpoint of being able to form a container that is less likely to break even when subjected to impacts such as dropping, propylene, 1-butene, 1-pentene, 1-hexene, 4-methyl-1-pentene, and 1-octene are preferred, and 1-butene, 1-pentene, 1-hexene, 4-methyl-1-pentene, and 1-octene are more preferred. The copolymer B may have only structural units derived from one of these α-olefins, or may have only structural units derived from a plurality of types.

[0020] The proportion of the structural unit derived from ethylene with respect to all the structural units of the copolymer B is preferably 70 mol% or more and 95 mol% or less, more preferably 75 mol% or more and 92 mol% or less, and even more preferably 80 mol% or more and 90 mol% or less. The proportion of the structural unit derived from an α-olefin having 3 to 20 carbon atoms with respect to all the structural units of the copolymer B is preferably 5 mol% or more and 30 mol% or less, more preferably 8 mol% or more and 25 mol% or less, and even more preferably 10 mol% or more and 20 mol% or less.

[0021] The density (ASTM D 1505) of the copolymer B is 3 850 kg / m 3 or more and 910 kg / m 3 or less, preferably 855 kg / m 3 or more and 905 kg / m 3 or less, more preferably 860 kg / m 3 or more and 890 kg / m 3 or less.

[0022] Copolymer B may be a random copolymer or a block copolymer. From the viewpoint of further increasing the flexibility of the heat seal layer and enabling the formation of a container that is more resistant to tearing even when subjected to impacts such as dropping, a random copolymer is preferred.

[0023] Copolymer C is a copolymer of propylene and an α-olefin (excluding propylene) having 2 to 20 carbon atoms. Examples of α-olefins other than propylene include ethylene, 1-butene, 1-pentene, 1-hexene, 4-methyl-1-pentene, 1-octene, 1-decene, 1-dodecene, 1-tetradecene, 1-hexadecene, 1-octadecene, and 1-eicosene. Of these, ethylene, 1-butene, 1-pentene, 1-hexene, 4-methyl-1-pentene, and 1-octene are preferred, and ethylene and 1-butene are more preferred, from the viewpoint of forming a container that is more resistant to tearing even when subjected to impact such as dropping. Copolymer C may have only structural units derived from one of these α-olefins, or it may have structural units derived from multiple types.

[0024] The proportion of structural units derived from propylene to the total structural units of copolymer C is preferably 51 mol% to 90 mol%, and more preferably 65 mol% to 80 mol%. The proportion of structural units derived from α-olefins (excluding propylene) with 2 to 20 carbon atoms to the total structural units of copolymer C is preferably 10 mol% to 49 mol%, and more preferably 11 mol% to 35 mol%.

[0025] The density of copolymer C (ASTM D 1505) is 860 kg / m³. 3 More than 899kg / m 3 Preferably, it is 860 kg / m 3 More than 895kg / m 3 It is more preferable that the following conditions are met: 865 kg / m 3 More than 890kg / m 3The following is even more preferable: Higher density tends to improve surface properties such as slipperiness and blocking properties. Also, lower density improves oxygen permeability and carbon dioxide permeability.

[0026] Furthermore, two or more propylene-based copolymers may be used as copolymer C.

[0027] Copolymer C may be a random copolymer or a block copolymer. From the viewpoint of further increasing the flexibility of the heat seal layer and enabling the formation of a container that is more resistant to tearing even when subjected to impacts such as dropping, a random copolymer is preferred.

[0028] The heat seal layer may contain either copolymer B or copolymer C, or both. From the viewpoint of improving tensile properties and reducing haze, it is preferable that the heat seal layer contains copolymer C.

[0029] Copolymers A, B, and C may form a sea-island structure in the heat-seal layer, with copolymer A forming the sea portion and copolymer B or C forming the island portion. When the container is subjected to impact, the sea-island structure causes localized fracture at the interface between the sea portion and the island portion, absorbing the stress caused by the impact. Therefore, the sea-island structure enhances the impact resistance of the heat-seal layer, enabling the formation of a container that is more resistant to tearing even when subjected to impacts such as drops. The presence or absence of a sea-island structure can be confirmed by staining a section cut from the multilayer film with osmium using a microtome or the like, and then observing it at a magnification of 10,000x using a transmission electron microscope (TEM).

[0030] The heat seal layer preferably contains copolymer B and copolymer C in amounts of 3% to 30% by mass relative to the total mass of copolymer A, copolymer B, and copolymer C. The higher the total content of copolymer B and copolymer C, the greater the flexibility of the heat seal layer, allowing for the formation of a container that is more resistant to tearing even when subjected to impacts such as drops. The lower the total content of copolymer B and copolymer C, the greater the transparency of the heat seal layer, and the greater the transparency of the resin film and the container formed therefrom. The total content of copolymer B and copolymer C relative to the total mass of copolymer A, copolymer B, and copolymer C is more preferably 5% to 25% by mass, and even more preferably 7% to 23% by mass. The content of copolymer A relative to the total mass of copolymer A, copolymer B, and copolymer C is preferably 70% to 97% by mass, more preferably 75% to 95% by mass, and even more preferably 77% to 93% by mass.

[0031] 1-1-2. Other resins The heat seal layer may contain resins other than copolymers A, B, and C. Examples of these other resins include ethylene homopolymers such as linear low-density polyethylene (LLDPE), propylene homopolymers, butene-based (co)polymers, and block copolymers of ethylene and silicone.

[0032] These resins can be used depending on the properties required for the multilayer film. For example, if it is desired to improve the antiblocking properties of the heat seal layer, the heat seal layer may contain a block copolymer of ethylene and silicone.

[0033] The above-mentioned block copolymer of ethylene and silicone can be a (polyethylene)-(silicone) binary block copolymer or a (polyethylene)-(silicone)-(polyethylene) ternary block copolymer, etc. The polyethylene may be a homopolymer of ethylene or a copolymer of ethylene and an α-olefin having 3 to 20 carbon atoms, but it is preferable that it be a homopolymer of ethylene. Furthermore, the molar ratio of ethylene to other α-olefins (ethylene:other α-olefins) when it is a copolymer is preferably 81:19 to 99:1, and more preferably 90:10 to 99:1.

[0034] For example, the block copolymer described above may have the following structure. A-CH2-CH2-Si(CH3)2-O-(Si(CH3)2-O) i -Si(CH3)2-CH2-CH2-A

[0035] Here, the two A's independently represent polyethylene, and i represents an integer of 1 or more. The polyethylene represented by each of the above A's preferably has a number-average molecular weight (Mn) of 100 to 500,000, more preferably 500 to 50,000, and even more preferably 700 to 10,000. i is preferably 1 to 1000, more preferably 1 to 300, and even more preferably 1 to 50.

[0036] The content of the above-mentioned other resins (for example, a block copolymer of ethylene and silicone) is preferably more than 0% by mass and 8% by mass or less, more preferably 0.5% by mass or more and 6% by mass or less, and even more preferably 1% by mass or more and 3% by mass or less, based on the total mass of the heat seal layer.

[0037] Ethylene-based (co)polymers, propylene-based (co)polymers, 4-methyl-1-pentene-based copolymers, and butene-based (co)polymers can be produced by polymerizing monomers in the presence of known catalysts such as Ziegler-Natta catalysts and metallocene catalysts using known polymerization methods such as gas-phase, bulk, and slurry methods. The monomers may be derived from biomass or fossil fuels. Alternatively, both biomass-derived and fossil fuel-derived monomers may be used.

[0038] 1-2. Core Layer The core layer is a layer that ensures the flatness of the multilayer film. The core layer may be located adjacent to the heat seal layer, or it may be located between the heat seal layer and the core layer with another layer in between.

[0039] The core layer contains a 4-methyl-1-pentene (co)polymer (hereinafter simply referred to as "(co)polymer D").

[0040] The (co)polymer D may be a homopolymer of 4-methyl-1-pentene or a copolymer of 4-methyl-1-pentene and another monomer. When it is a copolymer, the (co)polymer D is preferably a copolymer of 4-methyl-1-pentene and an α-olefin having 3 to 20 carbon atoms other than 4-methyl-1-pentene, and more preferably a copolymer of 4-methyl-1-pentene and a linear α-olefin having 3 to 20 carbon atoms. Examples of linear α-olefins having 3 to 20 carbon atoms include propylene, 1-butene, 1-pentene, 1-hexene, 1-octene, 1-decene, 1-dodecene, 1-tetradecene, 1-hexadecene, 1-octadecene, and 1-eicosene. Of these, a copolymer of 4-methyl-1-pentene and 1-hexadecene or 1-octadecene is preferred.

[0041] When it is a copolymer, the proportion of structural units derived from 4-methyl-1-pentene to the total structural units of (co)polymer D is preferably 80 mol% to 99 mol%. The proportion of structural units derived from ethylene or α-olefins other than 4-methyl-1-pentene to the total structural units of the copolymer (co)polymer D is preferably 1 mol% to 20 mol%. From the viewpoint of improving the oxygen permeability of the multilayer film, it is preferable that (co)polymer D has a high proportion of structural units derived from 4-methyl-1-pentene.

[0042] The (co)polymer D may be a resin consisting of the same structural units as copolymer A, but it is preferable that it is a resin containing different structural units.

[0043] The core layer may contain resins other than (co)polymer D. Examples of the other resins mentioned above include ethylene homopolymers such as linear low-density polyethylene (LLDPE), copolymers of ethylene and α-olefins having 3 to 20 carbon atoms, propylene homopolymers, copolymers of propylene and α-olefins (excluding propylene) having 2 to 20 carbon atoms, butene-based (co)polymers, and styrene-based elastomers.

[0044] These resins can be used depending on the properties required for the multilayer film. For example, from the viewpoint of forming a container that is more resistant to tearing even when subjected to impacts such as drops, and from the viewpoint of improving tensile properties, the core layer may contain a copolymer of ethylene and an α-olefin having 3 to 20 carbon atoms, or a copolymer of propylene and an α-olefin (excluding propylene) having 2 to 20 carbon atoms. Of these, from the viewpoint of increasing film impact strength and lowering haze, it is preferable that the core layer contains a copolymer of propylene and an α-olefin (excluding propylene) having 2 to 20 carbon atoms. The copolymer of ethylene and an α-olefin having 3 to 20 carbon atoms may be a resin consisting of the same structural units as copolymer B described for the heat seal layer, or it may be a resin containing different structural units. Furthermore, the copolymer of propylene and an α-olefin (excluding propylene) having 2 to 20 carbon atoms may be a resin consisting of the same structural units as copolymer C described for the heat seal layer, or it may be a resin containing different structural units.

[0045] Ethylene-based (co)polymers, propylene-based (co)polymers, 4-methyl-1-pentene-based copolymers, and butene-based (co)polymers can be produced by polymerizing monomers in the presence of known catalysts such as Ziegler-Natta catalysts and metallocene catalysts using known polymerization methods such as gas-phase, bulk, and slurry methods. The monomers may be derived from biomass or fossil fuels. Alternatively, both biomass-derived and fossil fuel-derived monomers may be used.

[0046] The core layer preferably contains, in an amount of 3% to 40% by mass of ethylene and an α-olefin having 3 to 20 carbon atoms, or a copolymer of propylene and an α-olefin (excluding propylene) having 2 to 20 carbon atoms, based on its total mass. The higher the copolymer content, the more resistant the container is to tearing even when subjected to impacts such as dropping. Furthermore, the higher the copolymer content, the higher the tensile properties of the multilayer film can be. The lower the copolymer content, the higher the oxygen permeability of the multilayer film can be. The copolymer content is more preferably 5% to 30% by mass, and even more preferably 7% to 25% by mass. The content of (co)polymer D relative to the total mass of the core layer is preferably 60% to 97% by mass, more preferably 70% to 95% by mass, and even more preferably 75% to 93% by mass.

[0047] 1-3. Characteristics of multilayer films The multilayer film according to this embodiment has an oxygen permeability of 3.0 L / (m²) at 23°C. 2 ·day · atm) or more 100.0L / (m 2 (·day·atm) or less, 4.0L / (m 2 ·day · atm) or more 50.0L / (m 2 It is preferable that it be less than or equal to 5.0 L / (m³) 2 ·day · atm) or more 30.0L / (m 2 It is more preferable that the temperature is below (day·atm). Increasing the oxygen permeability of the multilayer film can improve the efficiency of cell culture using cell containers.

[0048] Furthermore, the multilayer film has a carbon dioxide transmission rate of 10 L / (m²) at 23°C. 2 ·day · atm) or more 400L / (m 2 It is preferable that it is less than or equal to 10 L / (m³) 2 ·day · atm) or more 300L / (m 2It is preferable that the carbon dioxide permeability is less than or equal to (day·atm). The higher the carbon dioxide permeability, the more efficient the cell culture using the cell vessel can be.

[0049] Oxygen and carbon dioxide permeability were measured in accordance with JIS K 7126-1:2006 using a differential pressure gas permeability measuring device (manufactured by Toyo Seiki Seisakusho) at a test temperature of 23°C and a test relative humidity of 0%RH, with a measurement area of ​​5 cm² of multilayer film. 2 The measurement is performed as follows: When high oxygen or carbon dioxide permeability is expected, an aluminum mask is applied to the sample beforehand, and the actual permeable area is 5.0 cm². 2 It is preferable to do so.

[0050] The multilayer film has a film impact strength measured from the opposite side of the heat-seal layer in accordance with ASTM-D 3420:2021, which is between 7 kJ / m and 40 kJ / m, preferably 8 kJ / m or higher, and more preferably 9 kJ / m or higher. The higher the film impact strength, the more resistant the container is to tearing even when subjected to impacts such as dropping. There is no particular upper limit to the film impact strength, but it can be, for example, 40 kJ / m or lower.

[0051] The multilayer film preferably has a Young's modulus of 100 MPa to 500 MPa at 23°C, measured in accordance with JIS K 6781:1994, more preferably 150 MPa to 450 MPa, and even more preferably 200 MPa to 400 MPa. The lower the Young's modulus, the easier the multilayer film is to stretch and absorb energy from impact. There is no particular lower limit to the Young's modulus, but from the viewpoint of improving the transportability of the film, it is preferable to set it to 100 MPa or higher.

[0052] The multilayer film preferably has a tensile elongation at 23°C measured in accordance with JIS K 6781:1994 of 300% to 1000%, more preferably 320% to 1000%, and even more preferably 350% to 1000%. The greater the tensile elongation at 23°C, the easier the multilayer film is to stretch and absorb energy from impact. There is no particular upper limit to the tensile elongation at 23°C, but from the viewpoint of improving the cutability of the film, it is preferable to keep it at 1000% or less.

[0053] The multilayer film preferably has a tensile breaking strength at 23°C measured in accordance with JIS K 6781:1994 of 24 MPa to 100 MPa, more preferably 26 MPa to 100 MPa, and even more preferably 28 MPa to 100 MPa. The higher the tensile breaking strength, the less likely the multilayer film is to break and the better it absorbs energy from impact. There is no particular upper limit to the tensile elongation at break, but from the viewpoint of improving the cuttability of the film, it is preferable to keep it at 100 MPa or less.

[0054] The Young's modulus, tensile elongation at break, and tensile strength are the arithmetic mean values ​​obtained by measuring the multilayer film in the MD direction and TD direction (specifically, the long side direction and the short side direction).

[0055] It is preferable that the multilayer film has high heat sealing strength. Specifically, it is preferable that the heat sealing strength of the multilayer film is 5N / 15mm width or more and 300N / 15mm width or less, more preferably 10N / 15mm width or more and 200N / 15mm width or less, and even more preferably 20N / 15mm width or more and 200N / 15mm width or less.

[0056] When measuring the heat-sealing strength of a multilayer film, two rectangular heat-sealing test pieces measuring 150 mm wide x 50 mm high are cut from the resin film so that the vertical direction coincides with the MD (Machine Direction) direction of the resin film. Next, these two heat-sealing test pieces are placed on top of each other so that the heat-sealing layers of the two multilayer films face each other. Then, using a heat-sealing test machine (Tester Industries Co., Ltd., thermal gradient heat-sealing tester, model TP-701-G), the upper and lower temperatures (heat-sealing temperature) of the heat-sealing bar are set to 150°C, the seal width is 5 mm, the seal pressure is 0.2 MPa, and the seal time is 2 seconds, and the two heat-sealed multilayer films are removed from the test machine, and strip-shaped test pieces with a width of 15 mm are cut out in directions perpendicular and parallel to the heat-sealing line as surface-to-surface heat-sealing test pieces of the multilayer film. Using a tensile testing machine (Orientec Co., Ltd., Tensilon Universal Material Testing Machine, Model RTG-1250), strip-shaped test pieces were peeled at a test temperature of 23°C, with a chuck distance of 50 mm and a tensile speed of 300 mm / min. The maximum peel strength was measured and defined as the heat seal strength (unit: N / 15 mm). The heat seal strength was measured for five test pieces, and the average value was calculated.

[0057] Furthermore, the multilayer film has a moisture permeability of 10 g / m² at 40°C and 90% relative humidity. 2 ·day) or more 100g / (m 2 It is preferable that it is less than or equal to 12 g / (m 2 ·day) or more than 80g / (m 2 It is more preferable that it be less than or equal to 14g / (m 2 ·day) or more 60g / (m 2 It is even more preferable that the moisture permeability is less than or equal to (day). By keeping the moisture permeability within this range, moisture loss from the contents is minimized.

[0058] The moisture permeability is calculated according to the isobaric method (cup type - gravimetric method) described in JIS Z 0208:2021, under condition B (test temperature 40°C, test relative humidity 90%RH).

[0059] From the viewpoint of improving the efficiency of cell observation, the multilayer film is preferably highly transparent to visible light and has low haze. Specifically, the total light transmittance measured in accordance with ASTM D-1003:2021 is preferably 50% to 100%, more preferably 70% to 100%, and even more preferably 90% to 100%. In addition, the haze measured in accordance with ASTM D-1003:2021 is preferably 0% to 20%, and more preferably 0% to 10%.

[0060] Furthermore, the multilayer film has a sterilization assurance level (SAL) of 10, measured in accordance with BS EN556-1:2001 for medical devices. -3 Preferably, 10 -6 The following is more preferable. Here, SAL represents the degree of sterility of the sterilized items after the sterilization process, and is expressed as the probability of microorganisms being present per unit number of sterilized items after sterilization. SAL is 10 -n This is expressed as, and the number of viable bacteria per sterilized item is 10 -n This means that n is 3 or greater, and more preferably 6 or greater.

[0061] The multilayer film is preferably 50 μm to 1 mm thick, more preferably 50 μm to 500 μm thick, and even more preferably 50 μm to 300 μm thick. The thicker the multilayer film, the greater the strength of the container and the welding strength when heat-sealed. The thinner the multilayer film, the greater the permeability of oxygen and carbon dioxide gases.

[0062] The ratio of the thickness of the heat seal layer to the thickness of the core layer (heat seal layer / core layer) is preferably 1 / 10 or more and 5 / 1 or less, more preferably 1 / 7 or more and 3 / 1 or less, and even more preferably 1 / 4 or more and 2 / 1 or less. Increasing the ratio of the heat seal layer thickness can improve the heat seal strength and tensile properties. Increasing the ratio of the core layer thickness can reduce haze.

[0063] The thickness of the heat seal layer is preferably 5 μm to 300 μm, more preferably 5 μm to 200 μm, and even more preferably 5 μm to 150 μm. The thicker the heat seal layer, the better the heat sealability of the multilayer film. The thinner the heat seal layer, the better the oxygen permeability of the multilayer film.

[0064] The thickness of the core layer is preferably 10 μm to 500 μm, more preferably 15 μm to 300 μm, and even more preferably 20 μm to 100 μm. The thicker the core layer, the more the flatness of the multilayer film can be improved. By making the core layer appropriately thin, the oxygen permeability of the multilayer film can be improved.

[0065] Furthermore, the ratio of the thickness of the skin layer to the thickness of the core layer, as well as the thickness of the skin layer itself, can be the same as the ratio of the thickness of the heat seal layer to the thickness of the core layer and the thickness of the heat seal layer described above.

[0066] Furthermore, it is preferable that the multilayer film has an indicator portion indicating that the surface is a heat-seal layer, or an indicator portion indicating that the surface is a skin layer that is the outside of the cell container. The indicator portion may be formed by attaching a sticker to one or both surfaces of the multilayer film indicating which surface it is. Alternatively, the indicator portion may be formed by writing which surface it is with ink or the like on the protective layer described above.

[0067] 1-4. Method for manufacturing multilayer films The multilayer film described above can be manufactured by a process of preparing the materials for each layer and a process of melting the prepared materials and co-extruding them to obtain the multilayer film.

[0068] In the preparation process, a heat-seal layer material containing copolymer A and copolymer B or copolymer C, and a core layer material containing (co)polymer D are prepared. The materials for each of these layers can be any of the materials described above, and it is preferable to select them so that they satisfy the above-described properties after molding. In addition, if necessary, a skin layer material made of the same material as the heat-seal layer may be prepared.

[0069] In the process of obtaining a multilayer film, the materials for each layer are melted and kneaded, and then co-extruded using a T-die extrusion machine or an extrusion lamination machine. The co-extruded material may be molded by an inflation method or a casting method.

[0070] 2. Container Multilayer films can be used to create cell containers by stacking two or more multilayer films, or by folding a single multilayer film, overlapping the edges, and then heat-sealing the edges to form a bag. When stacking the films, the heat-sealed layers should be positioned facing each other.

[0071] Figure 1A is a plan view showing the appearance of a cell vessel manufactured from the multilayer film described above, and Figure 1B is a partial cross-sectional view of the cell vessel shown in Figure 1A along the dashed line 1B-1B.

[0072] The cell container 100 includes containers that are shaped like bags and are used to culture cells by introducing a culture medium and cells into them. In this specification, cell culture means to proliferate, grow, or maintain the cells in a living state.

[0073] The cell container 100 is formed by overlapping two multilayer films 112 and 114, and creating a sealed portion 116 around the entire circumference of the edges to form a substantially sealed bag portion 110. In a portion of the heat-sealed sealed portion 116, one or more port members 120 (three in this embodiment) are sandwiched between the multilayer films 112 and 114. The port members 120 are cylindrical members that connect the inside and outside of the bag portion 110, and are welded in contact with the heat-seal layer to adhere to these multilayer films. Alternatively, the port portion may be welded between the port member 120 and the heat-seal layer of the multilayer films 112 and 114 via another welding sheet.

[0074] The heat sealing can be performed, for example, at a temperature of 120°C to 200°C, preferably 140°C to 180°C, and at a pressure of 0.1 MPa to 0.5 MPa, preferably 0.1 MPa to 0.2 MPa, for 1 second to 10 seconds, preferably 1 second to 5 seconds.

[0075] The heat sealing described above joins the heat-sealed layers of the stacked multilayer films together.

[0076] Furthermore, when stacking and arranging multilayer films, the multilayer films may be shaped into a predetermined form by methods such as vacuum forming, pressure forming, and vacuum pressure forming. In this case as well, it is sufficient that the edges of the multilayer films are stacked and arranged. As for the joining method of multilayer films, in addition to the method of heat sealing by sandwiching the stacked multilayer films between two hot plates, high-frequency welding and laser welding can also be applied. For example, in the case of laser welding, as described in the method of Japanese Patent No. 4279674, a method can be used in which laser light in the wavelength range of 1.8 to 2 μm emitted from a Ho-YAG laser or Tm fiber laser, or laser light with a wavelength of 10.6 μm emitted from a carbon dioxide laser is irradiated onto the parts to be joined of the stacked multilayer films, causing the multilayer films to directly absorb the energy of the laser light and melt the parts to be joined, thereby welding them together.

[0077] Furthermore, the number of multilayer films used to prepare the cell container 100 is not limited to two. One multilayer film may be folded, its edges overlapping, and the edges heat-sealed to form a bag, or three or more multilayer films may be used to form the cell container.

[0078] Furthermore, cell containers can be sterilized after preparation and before use. Gamma ray sterilization is preferred as the sterilization method, given the gamma-ray irradiation resistance of the film material. The gamma ray irradiation dose is preferably in the range of 10 to 50 kGy. Additionally, cell containers can preferably be used as single-use items to prevent contamination and maintain sterility.

[0079] 3. Methods for culturing cells The cell vessels described above can be used for culturing various types of cells.

[0080] Specifically, an injection port for injecting drugs or other substances, and a tube for connecting to a Luer port are attached to each port component 200. Then, a culture medium containing cells (cell suspension) is introduced into the cell container 100 through the Luer port connected to the tube. Alternatively, a culture medium without cells may be introduced into the cell container 100 first, and then the cell suspension may be introduced afterward.

[0081] Subsequently, the cell container 100 is placed in the incubator and the cells are cultured. The conditions for culturing the cells are not particularly limited and should be selected according to the type of cells being cultured.

[0082] During culture, drugs may be injected through the injection port, or a portion of the cells may be taken to check the culture status. Additionally, some or all of the cells may be transferred to another cell container 100 via a tube attached to the port component.

[0083] Finally, the cultured cells are collected through the tube attached to the port component.

[0084] The cells targeted by the cell container 100 are not particularly limited and may be differentiated somatic cells or undifferentiated stem cells. The cells may be living cells or dead cells.

[0085] 4. Other Embodiments It should be noted that each of the embodiments described above is merely an example of the present invention, and the present invention is not limited to the embodiments described above. It goes without saying that many other diverse embodiments are possible within the scope of the concept of the present invention.

[0086] For example, the multilayer film mentioned above can be used not only for cell containers but also for cell cryopreservation containers, and so on. [Examples]

[0087] The present invention will be described in detail based on examples, but the present invention is not limited to these examples.

[0088] 1. Fabrication of multilayer and single-layer films 1-1.Materials The following materials were prepared. • 4-methyl-1-pentene copolymer 1 (4MP1-1) A 4-methyl-1-pentene copolymer (melting point: 130°C) having a composition of 85 mol% of constituent units derived from 4-methyl-1-pentene and 15 mol% of constituent units derived from propylene was designated as 4MP1-1 (Copolymer A). • 4-methyl-1-pentene copolymer 2 (4MP1-2) A 4-methyl-1-pentene copolymer (melting point: 224°C) was defined as 4MP1-2, in which the proportion of constituent units derived from 4-methyl-1-pentene was 97.6 mol%, the proportion of constituent units derived from 1-hexadecene was 1.44 mol%, and the proportion of constituent units derived from 1-octadecene was 0.96 mol%. • 4-methyl-1-pentene copolymer 3 (4MP1-3) A 4-methyl-1-pentene copolymer having a composition of 72 mol% derived from 4-methyl-1-pentene and 28 mol% derived from propylene was designated as 4MP1-3. • Copolymer of propylene and α-olefin (PP) A propylene-based resin composition (MFR (ASTM D1238), temperature 230°C, load 2.16kg) 6.0g / 10min, density 868kg / m³) is obtained by melt-kneading 90% by mass of a propylene-ethylene-1-butene copolymer (Copolymer C), which is prepared according to the method described in the Examples section of the Third Invention in International Publication No. 2006 / 57361, has an ethylene content of 16 mol%, a propylene content of 78 mol%, a 1-butene content of 6 mol%, and an MFR (according to ASTM D1238, temperature 230°C, load 2.16kg) of 6g / 10min, and 10% by mass of a propylene homopolymer (MFR (ASTM D1238, temperature 230°C, load 2.16kg) 7.0g / 10min, melting point 160°C). 3 ) was used as a propylene copolymer (PP). • Copolymer of ethylene and α-olefin (PE) An ethylene copolymer in which 89 mol% of constituent units are derived from ethylene and 11 mol% of constituent units are derived from 1-butene (MFR (ASTM D1238 compliant, temperature 230°C, load 2.16 kg) 6.7 g / 10 min, density 885 kg / m³) 3 The ethylene copolymer (PE) (Copolymer B), with a melting point of 66°C, was used. • Linear low-density polyethylene (LLDPE) Evolu SP2040 (melting point: 116℃), manufactured by Prime Polymer Co., Ltd., was used as LLDPE. • Copolymer of butene and α-olefin (PB) An ethylene copolymer (compliant with MFR ASTM D1238, temperature 230°C, load 2.16 kg) with a composition of 98 mol% 1-butene-derived units and 2 mol% ethylene-derived units, 9.0 g / 10 min, density 910 kg / m³ 3 A butene copolymer (PB) with a melting point of 100°C was used. • Block copolymer of ethylene and silicone Polyethylene (P'-1) having a vinyl group at one end, synthesized according to the method described in Synthesis Example 2 of International Publication No. 2012 / 098865, was used as an ethylene / silicone block copolymer.

[0089] 1-2. Fabrication of multilayer and single-layer films Using a tumbler blender, each material was mixed (dry blended) in the proportions shown in Table 1 to prepare resin compositions for each layer.

[0090] Each layer's resin composition was supplied to its respective extruder, and using a cast molding die (die width 350 mmφ, lip gap 1 mm), the extrusion rate of each extruder was set so that the resin temperature reached 270°C and the thickness ratio of the skin layer, core layer, and heat seal layer was 1:1:1 or 1:2:1 in that order. Multilayer films 1 to 10 with a thickness of 100 μm were obtained by co-extrusion molding. The molding speed was 4 m / min.

[0091] LLDPE was supplied to an extruder, and a single-layer film 11 with a thickness of 100 μm was obtained by single-layer extrusion molding using a cast molding die (die width 350 mmφ, lip gap 1 mm) at a resin temperature of 230°C. The molding speed was 4 m / min.

[0092] Tables 1 and 2 show the total thickness, thickness ratio, and materials and their quantities used in each layer of the fabricated multilayer films 1 to 10 and single-layer film 11. In Tables 1 and 2, the "thickness ratio" column indicates the ratio of the skin layer / core layer / heat seal layer thicknesses, and the "materials for each layer" column indicates the quantity (in parts by mass) of each material used in the fabrication of each layer.

[0093] [Table 1]

[0094] [Table 2]

[0095] 2. Measurement of multilayer and single-layer films 2-1. Film Impact Strength Using a film impact tester manufactured by Toyo Seiki Seisakusho Co., Ltd. (compliant with ASTM-D 3420:2021), the impact strength of each film was measured from the skin layer side, with a film size of 100mm x 100mm, an impact head spherical shape of 0.5 inches in diameter, and a measurement temperature of 23°C.

[0096] 2-2. Oxygen permeability Oxygen permeability was measured in accordance with JIS K 7126-1:2006 using a differential pressure gas permeability measuring device (manufactured by Toyo Seiki Seisakusho) under test conditions of a test temperature of 23°C and test humidity of 0%RH, with a measurement area of ​​5 cm² of film. 2 The measurement was performed as follows: The measurement area of ​​the film was adjusted by preparing two adhesive aluminum masks manufactured by Modern Control, each with a 25mm diameter hole in the center, and stacking the film to be measured between these two masks. Specifically, the film was positioned so that the central holes of the two masks overlapped.

[0097] 2-3. Tensile Test For each multilayer and single-layer film, test specimens were cut into strips measuring 15 mm wide x 100 mm long. In accordance with JIS K7127:1999, a tensile testing machine (Instron, universal tensile testing machine 3380) was used to measure Young's modulus (YM) (in MPa), tensile elongation at break (EL) (in %), and tensile strength at break (TS) (in MPa) in the MD and TD directions of the test specimens under the conditions of a chuck distance of 50 mm, a tensile speed of 300 mm / min, and a temperature of 23°C. Measurements were performed for both the MD and TD directions, and the average value of these measurements was taken as the measurement value for the film in question.

[0098] 2-4. Heat seal strength Two strips measuring 150 mm wide x 50 mm long were prepared from each of the multilayer and single-layer films to serve as test specimens. Next, the two prepared test specimens were stacked so that the heat-seal layers faced each other, and then heat-sealed using a heat-seal tester (Tester Industries Co., Ltd., Thermal Gradient Heat Seal Tester TP-701-G) under the following conditions: upper temperature 150°C, lower temperature 150°C, seal width 5 mm, seal pressure 0.2 MPa, and seal time 2 seconds.

[0099] Next, the heat-sealed test specimens were removed from the heat-seal testing machine and cut into 15mm wide strips. These 15mm wide heat-sealed test specimens were then subjected to a test at a speed of 300mm / min and an ambient temperature of 23°C. The specimens were pulled at a 180° angle to the heat-sealed surface between them to separate them, and the maximum peel strength was measured. This maximum value was defined as the heat-seal strength (unit: N / 15mm). If the specimens stretched to their limit without separation, the maximum observed tensile strength was defined as the heat-seal strength. The heat-seal strength was measured for five specimens, and the average value was calculated.

[0100] 2-5. Haze and total light transmittance Each multilayer and single-layer film was cut into 50mm squares to form test specimens. The haze value (in %) and total transmitted light in air were measured using a fully automatic haze meter (Tokyo Denshoku Co., Ltd., TC-HIII DPK, light source: 12V 50W halogen lamp C) in accordance with ASTM D 1003:2021. This measurement was performed at three arbitrary points on each test specimen, and the average of the data from these three points was taken as the measured value. The total light transmittance was then calculated using the following formula. Total light transmittance (%) = 100 × (total transmitted light amount) / (incident light amount)

[0101] 2-6. Moisture permeability The moisture permeability was calculated under condition B (test temperature 40°C, test humidity 90%RH) in accordance with the isobaric method (cup type - gravimetric method) described in JIS Z 0208:2021.

[0102] 2-7.Sea-island structure For multilayer films 1-10 and single-layer film 11, 100 μm thick sample pieces were prepared using a microtome. The cut sample pieces were stained with osmium and observed at a magnification of 10,000x using a transmission electron microscope (TEM) to confirm the presence or absence of sea-island structures in the heat-sealed layers.

[0103] The measurement results for multilayer films 1-10 and single-layer film 11 are shown in Tables 3 and 4.

[0104] [Table 3]

[0105] [Table 4]

[0106] 3. Preparation and evaluation of cell culture bags 3-1. Drop test of cell culture bags Two films measuring 210 mm in length and 148 mm in width, cut from each multilayer film, were prepared. They were placed so that the heat-seal edges faced each other, and the three sides were heat-sealed with a 10 mm width at a sealing temperature of 150°C, a sealing pressure of 0.2 MPa, and a sealing time of 1 second to obtain a bag component.

[0107] High-density polyethylene (melting point 130°C, density 960 kg / m³) is produced by injection molding. 3Port members were fabricated using either MFR 190℃·2.16kgf=13g / 10min or 4MP1-2. The fabricated port members had circular cross-sections in both the narrow and wide sections, with an outer diameter of 8mm and an inner diameter of 4mm for the narrow section, an outer diameter of 10mm for the wide section, and an inner diameter of 6mm for the wide section. Bags were fabricated using port members made of high-density polyethylene for single-layer films 11, and using port members made of 4MP1-2 for other multi-layer films.

[0108] A cell culture bag with a port component was obtained by heat sealing the opening of the bag component with a sealing temperature of 220°C, a sealing pressure of 0.2 MPa, and a sealing time of 5 seconds, with a weld line width (joint width) of 10 mm.

[0109] The resulting cell culture bags with port components were filled with 300 ml of water, and the port components were sealed with a silicone resin stopper with an upper diameter of 13 mm, a lower diameter of 10 mm, and a height of 18 mm. After standing for 1 day in an atmosphere of 23°C, the water-filled bags were dropped horizontally from a height of 50 cm (so that the flat side of the bag hit the surface of impact) in accordance with JIS Z 0238:1998. If the bags did not rupture, they were then dropped vertically from a height of 50 cm (so that the heat-sealed part opposite the port component hit the surface of impact). The size of the rupture in the bags after dropping was measured, and they were classified according to the following classification criteria. ○: Does not rupture even with horizontal or vertical drops. Small: Damage area less than 3cm in size Medium: Damage area is 3cm or larger but less than 5cm in size. Large: Damage area is 5cm or larger.

[0110] Table 5 shows the evaluation results of cell culture bags made from each film.

[0111] [Table 5]

[0112] The results shown in Tables 1 to 5 indicate that multilayer films with a film impact strength of 7 kJ / m to 40 kJ / m can be used to form containers that are resistant to tearing even when dropped or subjected to other impacts while containing liquid.

[0113] 3-2. Cell culture evaluation using cell culture bags Two films measuring 210 mm in length and 148 mm in width, cut from a multilayer film, were prepared. They were positioned so that the heat-seal edges faced each other, and the three sides were heat-sealed with a 10 mm width at a sealing temperature of 150°C, a sealing pressure of 0.2 MPa, and a sealing time of 1 second to obtain a bag component.

[0114] 3-2-1. Preparation of cell culture bags (Cell culture bag 1) Two pieces of film measuring 120 mm in length and 120 mm in width, cut from multilayer film 4, were prepared and placed facing each other. The three sides were heat-sealed with a 10 mm width seal at a sealing temperature of 150°C and a sealing pressure of 0.1 MPa to obtain a bag component.

[0115] A port component made from 4MP1-2 was placed at the opening of the bag component, and a cell culture bag 1 with a port was obtained by heat sealing with a welding line width (joint width) of 10 mm under the conditions of a sealing temperature of 240°C, a sealing pressure of 0.1 MPa, and a sealing time of 4 seconds. The culture space area of ​​the cell culture bag 1 with a port is 100 cm². 2 That was the case.

[0116] (Cell culture bag 2) Two pieces of single-layer film 11, each measuring 120 mm in length and 120 mm in width, were prepared. They were placed facing each other, and the three sides were heat-sealed with a 10 mm width seal at a sealing temperature of 150°C and a sealing pressure of 0.1 MPa to obtain a bag component.

[0117] A port component made of high-density polyethylene was placed at the opening of the bag component, and a cell culture bag 2 with a port was obtained by heat sealing with a welding line width (joint width) of 10 mm under the conditions of a sealing temperature of 220°C, a sealing pressure of 0.1 MPa, and a sealing time of 4 seconds. The culture space area of ​​the cell culture bag 2 with a port is 100 cm². 2 That was the case.

[0118] (Cell culture bag 3) Two pieces of film measuring 100 mm in length and 50 mm in width were cut from multilayer film 4, placed facing each other, and heat-sealed on three sides with a 10 mm width at a sealing temperature of 150°C and a sealing pressure of 0.1 MPa to obtain a bag component.

[0119] A port component made from 4MP1-1 was placed at the opening of the bag component, and a cell culture bag 3 with a port was obtained by heat sealing with a welding line width (joint width) of 10 mm under the conditions of a sealing temperature of 240°C, a sealing pressure of 0.1 MPa, and a sealing time of 4 seconds. The culture space area of ​​the cell culture bag 3 with a port is 24 cm². 2 That was the case.

[0120] (Cell culture bag 4) Two pieces of film measuring 100 mm in length and 50 mm in width were cut from a single-layer film 11. These were placed facing each other, and the three sides were heat-sealed with a 10 mm width at a sealing temperature of 150°C and a sealing pressure of 0.1 MPa to obtain a bag component.

[0121] A port component made of high-density polyethylene was placed at the opening of the bag component, and a cell culture bag 4 with a port was obtained by heat sealing with a welding line width (joint width) of 10 mm under the conditions of a sealing temperature of 220°C, a sealing pressure of 0.1 MPa, and a sealing time of 4 seconds. The culture space area of ​​the cell culture bag 4 with a port is 24 cm². 2 That was the case.

[0122] Cell culture bags with ports were sterilized by irradiating them with gamma rays at a dose of 25 kGy. The Sterility Assurance Level (SAL) of medical devices, measured according to BS EN556-1:2001, for cell culture bags manufactured under the same conditions and sterilized with gamma rays at a dose of 25 kGy was 10 in all cases. -6 That was the case.

[0123] 3-2-2. Cell culture test [Test 1] 2 x 10 7 One human chronic myeloid leukemia cell line K562 (RIKEN BRC, RCB0027) was suspended in 100 ml of RPMI1640 medium (Fujifilm Wako Pure Chemical Industries) containing 10% FBS (BioWest), 1% GlutaMAX™-I supplement (Thermo Fisher Scientific), and 1% penicillin / streptomycin (Fujifilm Wako Pure Chemical Industries). The cells were introduced into the sterile ported cell culture bags 1 and 2 and cultured in a CO2 incubator maintained at 37°C and 5% CO2. The cell suspension was collected from the port using a syringe 3 and 7 days after the start of culture.

[0124] Cell density and cell viability were calculated for cell suspensions 3 and 7 days after the start of culture using the trypan blue efflux method. Specifically, the cell suspension was mixed with a 0.4 w / v% trypan blue solution (Fujifilm Wako Pure Chemical Industries, Ltd.), injected into a cell counter (Funakoshi Co., Ltd.), and observed using a phase-contrast microscope (OLYMPUS Co., Ltd.) to count the cell density. Cell viability was classified as follows: cells that did not stain due to trypan blue exclusion were classified as live cells, and cells stained blue with the dye were classified as dead cells. Cell viability was calculated using the formula: live cell density ÷ total cell (live cells + dead cells) density. The results are shown in Tables 6 and 7.

[0125] [Table 6]

[0126] [Table 7]

[0127] The results shown in Tables 6 and 7 indicate that cell culture bag 1 has a higher cell culture efficiency than cell culture bag 2.

[0128] [Exam 2] 3 x 10 6 Human peripheral blood mononuclear cell (PBMC) cells (LONZA) were suspended in 6 ml of RPMI1640 medium (Gibco) containing 10% FCS, 1% penicillin / streptomycin, 50 ng / ml CD3 monoclonal antibody (Thermo Fisher Scientific), 2 μg / ml CD28 monoclonal antibody (Thermo Fisher Scientific), and 500 IU / ml recombinant human interleukin 2 (proteintech), and introduced into the sterile cell culture bags 3 and 4 equipped with ports. The cell culture bags containing the cell suspensions were placed in a CO2 incubator maintained at 37°C and 5% CO2 and cultured for 20 days. The entire cell suspension was collected from the port using a syringe 3, 6, 9, 12, 15, and 17 days after the start of culture. The collected cell suspension was centrifuged at 400g for 10 minutes, and 3 ml of the supernatant was removed. 3 ml of RPMI1640 medium containing 10% FCS, 1% penicillin / streptomycin, and 500 IU / ml recombinant human interleukin 2 was added, the suspension was allowed to rise, and the cells were reintroduced into a cell culture bag.

[0129] Cell density and cell viability were calculated for cell suspensions 20 days after the start of culture using the trypan blue efflux method. Specifically, the cell suspension was mixed with a 0.4 w / v% trypan blue solution (Gibco), injected into a cell counter (Wakken B-Tech Co., Ltd.), and observed using a phase-contrast microscope (OLYMPUS) to count the cell density. Cell viability was classified as follows: cells that did not stain due to trypan blue efflux were classified as live cells, and cells stained blue with the dye were classified as dead cells. Cell viability was calculated using the formula: live cell density ÷ total cell (live cells + dead cells) density. The results are shown in Tables 10 and 11.

[0130] [Table 8]

[0131] [Table 9]

[0132] The results shown in Tables 8 and 9 indicate that cell culture bag 3 has a higher cell culture efficiency than cell bag 4. [Industrial applicability]

[0133] The multilayer film according to the present invention and the cell container having the same can be applied to the culture of various cells. [Explanation of symbols]

[0134] 100 cell containers 110 Bag section 112, 114 multilayer film 116 Contact area 120 Port Components

Claims

1. A heat seal layer comprising copolymer A, which is a copolymer of 4-methyl-1-pentene and propylene, copolymer B, which is a copolymer of ethylene and an α-olefin having 3 to 20 carbon atoms, or copolymer C, which is a copolymer of propylene and an α-olefin (excluding propylene) having 2 to 20 carbon atoms, A core layer comprising a (co)polymer D which is a 4-methyl-1-pentene (co)polymer, The film impact strength measured from the opposite side of the heat-seal layer in accordance with ASTM-D 3420:2021 is 7 kJ / m or more. The oxygen permeability at 23°C is 3.0 L / (m³). ２ ・24h・atm) or more 100.0L / (m ２ - 24 hours (atm) or less Multilayer film.

2. The Young's modulus at 23°C, as measured in accordance with JIS K 6781:1994, is between 100 MPa and 500 MPa. The multilayer film according to claim 1.

3. The tensile elongation at 23°C, measured in accordance with JIS K 6781:1994, is between 300% and 1000%. The multilayer film according to claim 1.

4. The haze, as measured in accordance with ASTM D-1003:2021, is between 0% and 40%. The multilayer film according to claim 1.

5. The thickness is 50 μm or more and 1 mm or less. The multilayer film according to claim 1.

6. The ratio of the thickness of the heat seal layer to the thickness of the core layer (heat seal layer / core layer) is between 1 / 10 and 5 / 1. The multilayer film according to claim 1.

7. The sterility assurance level (SAL) of the medical device, as measured in accordance with BS EN556-1:2001, is 10. －３ The following is: The multilayer film according to claim 1.

8. The heat seal strength when the heat seal layers are heat-sealed together at 150°C is 10 N / 15 mm or more and 300 N / 15 mm or less. The multilayer film according to claim 1.

9. The heat seal layer has a sea-island structure. The multilayer film according to claim 1.

10. The copolymer A comprises 70 mol% to 99 mol% of 4-methyl-1-pentene and 1 mol% to 30 mol% of propylene relative to the total number of constituent units. The multilayer film according to claim 1.

11. The heat seal layer contains copolymer B and copolymer C in an amount of 3% to 30% by mass relative to the total mass of copolymer A, copolymer B, and copolymer C. The multilayer film according to claim 1.

12. The core layer comprises a copolymer of ethylene and an α-olefin having 3 to 20 carbon atoms, or a copolymer of propylene and an α-olefin (excluding propylene) having 2 to 20 carbon atoms. The multilayer film according to claim 1.

13. Used in the formation of cell vessels, The multilayer film according to claim 1.

14. One or more multilayer films according to any one of claims 1 to 13 are heat-sealed together to form a bag, container.

15. The container has a port member that connects the inside and outside of the container, The port member is bonded to the multilayer film in the heat-sealed portion of the heat-seal layer. The container according to claim 14.

16. The container according to claim 14, which is a cell container.

17. A method for manufacturing a container, comprising the step of heat-sealing one or more multilayer films according to any one of claims 1 to 11 to form a bag.

18. A step of introducing cells into a cell container comprising a multilayer film according to any one of claims 1 to 11, The process of culturing cells in the aforementioned cell vessel, A method for culturing cells, comprising the following characteristics.

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