Gas permeable material

Abstract

The invention relates to a gas permeable material. Gas permeable fluoropolymers and silicone materials for use in the construction of cell culture bags are described.

Description

[0001] This application is a divisional application of the invention patent application with application date of December 22, 2015, application number 201580069838.9, and name “GAS PERMEABLE MATERIAL”.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This patent application claims the benefit under 35 U.S.C. §119(e) of U.S. Serial No. 62 / 095,116, filed on December 22, 2014, entitled “FLUORINATED SILICONE FILM FOR OXYGEN PERMEABLE MATERIAL,” and U.S. Serial No. 14 / 976,372, filed on December 21, 2015, the contents of which are incorporated herein by reference in their entirety. Technical Field

[0004] The present invention generally relates to gas permeable materials and their use in cell culture apparatus and cell culture methods. Background Art

[0005] In vitro cell culture is a complex process by which cells are grown under controlled conditions, generally outside of their natural environment, but as close as possible to their natural in vivo conditions. In practice, cell culture refers to the culture of cells derived from multicellular eukaryotic organisms, especially animal cells. However, there are also cultures of plants, fungi, insects, and microorganisms, including viruses, bacteria, and protists.

[0006] In vitro cell culture provides essential materials for research and applications in pharmacology, physiology and toxicology. This includes bioprocessing and cell therapy when cell culture is necessary.

[0007] Cells are grown and maintained in a cell culture incubator at a suitable temperature and gas mixture. Typically, mammalian cells are incubated at 37°C with the pH maintained between 7.2 and 7.4. The pH is typically controlled using a bicarbonate buffer system in the culture medium together with an incubator atmosphere of approximately 5-7% carbon dioxide by volume. Carbon dioxide reacts with water to form carbonic acid, which in turn interacts with bicarbonate ions in the culture medium to form a buffer system that maintains the pH near physiological levels. Oxygen is essential for cell metabolism and growth. Culture conditions are variable for each cell type, and for a particular cell type, changes in conditions can result in different phenotypes.

[0008] A variety of cell types are grown in culture, including connective tissue cells, bone cells, cardiac cells, epithelial cells, neural cells, endocrine cells, immune cells, lymphocytes, melanocytes, and many types of tumor cells. Similarly, a variety of culture media are available depending on the specific growth needs and growth conditions of the cells.

[0009] Commercially available gas permeable cell culture bags are currently the standard device format for cell culture. Cell culture bags constructed with gas permeable membranes advantageously provide a large surface area for gas exchange while maintaining a closed system. Disposable use also helps reduce the risk of cell culture and environmental contamination.

[0010] Cell culture bags from OriGen Biomedical Group (OriGen PermaLife TM Bags), Baxter (related to U.S. Patent Nos. 4,829,002, 4,937,194, 5,935,847, 6,297,046B1 X-Fold TM )、Medtronic(Si-Culture TM , U.S. Patent No. 5,686,304), Biovectra (VectraCell TM ), and American Fluoroseal (VueLife covered by U.S. Patent Nos. 4,847,462 and 4,945,203 TM Culture Bag System) is commercially available.

[0011] The gas permeable membrane should be selected based on a variety of properties including gas permeability, moisture vapor permeability, ability to be modified for desired cell-to-cell interactions, optical clarity, physical strength, etc. There is a wide variety of information describing the types of gas permeable materials that have been successfully used for cell culture.

[0012] Among various rubber materials, silicone rubber is preferred in most cases in terms of its electrical properties, low cost, precision moldability, and durability and release migration in repeated bending.

[0013] Silicone membranes for cell culture bags have high oxygen permeability, good optical clarity, good penetration resistance, generally do not bind cells, and can be easily made into a variety of shapes. In the surface area where gas transfer is required, the silicone membrane can be less than about 3 mm, about 2 mm, about 1 mm, or about 0.8 mm. The best choice of material depends on the application.

[0014] Fluoropolymer membranes have desirable properties that make them a popular choice for culture bags. Compared to silicone, fluoropolymer membranes are more biologically, chemically, and immunologically inert, and are hydrophobic. Fluoropolymer membranes such as FEP (fluorinated ethylene-propylene) do not induce immune responses in immune cells and ancestral immune cells. However, fluoropolymer membranes are more expensive, less gas permeable than silicone membranes, and difficult to adhere to other polymers.

[0015] Therefore, there is a need to mitigate the disadvantages of using silicone or fluoropolymers alone for cell culture bags. Summary of the invention

[0016] In one aspect, the present invention provides an oxygen permeable composition. In another aspect, the present invention provides a two-part composite material having a first elastomeric layer and a second fluid contact layer, wherein the second fluid contact layer has a molecular weight of less than 1 mg / cm 2 The second fluid contact layer having low TOC can be a fluoropolymer, and the first elastomeric layer and the second fluoropolymer layer are adhered together. More particularly, embodiments disclosed herein relate to fluoropolymer films capable of bonding to the surface of an elastomeric rubber. Fluoropolymer coatings exhibit high bond strength with or without the use of an adhesive.

[0017] In another embodiment, the outer elastomer layer is natural polyisoprene rubber (NR), synthetic polyisoprene rubber (IR), polybutadiene rubber (BR), chloroprene rubber (CR), butyl rubber (IIR), halogenated butyl rubber (CIIR, BIIR), styrene butadiene rubber (SBR), nitrile rubber (NBR) and hydrogenated nitrile rubber (HNBR), ethylene propylene rubber (EPM), ethylene propylene diene monomer rubber (EPDM), epichlorohydrin rubber (ECO), polyacrylate rubber (ACM, ABR), silicone rubber (SI, Q, VMQ), fluorosilicone rubber (FSR, FVMQ), fluoroelastomer (FKM, FEPM), perfluoroelastomer (FFKM), polyether block amide (PEBA), chlorosulfonated polyethylene (CSM), ethylene vinyl acetate (EVA), cyclic olefin copolymer, polyolefin elastomer, elastomer PET or a mixture thereof.

[0018] In another embodiment, the outer elastomeric layer is a polymethylpentene polymer (PMP).

[0019] In another embodiment, the oxygen permeable membrane includes an outer silicone rubber layer and an inner fluoropolymer layer, wherein the inner fluoropolymer layer is coated on the outer silicone rubber layer.

[0020] In another embodiment, the silicone rubber of the oxygen permeable membrane is high consistency rubber (HCR), fluorosilicone rubber (FSR), liquid silicone rubber (LSR), or room temperature vulcanized rubber (RTV), thermoplastic silicone rubber (TPE), platinum-cured silicone rubber or peroxide-cured silicone rubber.

[0021] In another embodiment, the silicone rubber of the oxygen permeable membrane is thermoset, cured, vulcanized, catalyzed or injection molded.

[0022] In another embodiment, the fluoropolymer layer of the membrane is fluorinated ethylene-propylene (FEP), polytetrafluoroethylene (PTFE), tetrafluoroethylene-perfluoro(propyl vinyl ether) (PFA), polyvinylidene fluoride, hexafluoropropylene / tetrafluoroethylene / vinylidene copolymer (THV) or perfluoro(1-butenyl vinyl ether) homocyclopolymer with functionalized polymer end groups.

[0023] In another embodiment, the elastomeric, eg, PMP or silicone, layer and the fluoropolymer layer of the oxygen permeable membrane are bonded.

[0024] In yet another embodiment, the bonding of the elastomeric (e.g., PMP or silicone) layer and the fluoropolymer layer of the oxygen permeable membrane is performed by coating, RF welding, ultrasonic welding, hot bar welding, chemical bonding, adhesive bonding, hot melt bonding, solvent welding, laser welding, corona discharge, radiation, lamination (e.g., by extreme heat, tape or melt lamination), etching, plasma treatment, wetting, adhesives, radiation, extrusion, coextrusion, or a combination thereof.

[0025] In another embodiment, an oxygen permeable container includes an outer elastomeric layer and an inner fluid contact layer having a low TOC. In one aspect, the fluid contact layer is a fluoropolymer layer, wherein the inner fluoropolymer layer is adhered to the outer elastomeric layer of the container.

[0026] In another embodiment, the outer elastomeric layer of the container is natural polyisoprene rubber (NR), synthetic polyisoprene rubber (IR), polybutadiene rubber (BR), chloroprene rubber (CR), butyl rubber (IIR), halogenated butyl rubber (CIIR, BIIR), styrene butadiene rubber (SBR), nitrile rubber (NBR) and hydrogenated nitrile rubber (HNBR), ethylene propylene rubber (EPM), ethylene propylene diene monomer rubber (EPDM), epichlorohydrin rubber (ECO), polyacrylate rubber (ACM, ABR), silicone rubber (SI, Q, VMQ), fluorosilicone rubber (FSR, FVMQ), fluoroelastomer (FKM, FEPM), perfluoroelastomer (FFKM), polyether block amide (PEBA), chlorosulfonated polyethylene (CSM), ethylene vinyl acetate (EVA), cyclic olefin copolymer, polyolefin elastomer, elastomer PET or a combination thereof.

[0027] In another embodiment, the outer elastomeric layer of the container is a polymethylpentene polymer (PMP).

[0028] In another embodiment, the outer elastomeric layer of the container is silicone rubber.

[0029] In yet another embodiment, the silicone rubber of the oxygen permeable container is high consistency rubber (HCR), fluorosilicone rubber (FSR), liquid silicone rubber (LSR), or room temperature vulcanized rubber (RTV), thermoplastic silicone rubber (TPE), platinum-cured silicone rubber, or peroxide-cured silicone rubber.

[0030] In yet another embodiment, the silicone rubber of the oxygen permeable container is thermoset, cured, vulcanized, catalyzed, or injection molded.

[0031] In another embodiment, the fluoropolymer layer of the container is fluorinated ethylene-propylene (FEP), polytetrafluoroethylene (PTFE), polyvinylidene fluoride (PVDF), tetrafluoroethylene-perfluoro(propyl vinyl ether) (PFA), polyvinylidene fluoride (PVF), polychlorotrifluoroethylene (PCTFE), tetrafluoroethylene / hexafluoropropylene / ethylene copolymer (HTE), chlorotrifluoroethylene / vinylidene fluoride copolymer, chlorotrifluoroethylene / hexafluoropropylene, ethylene / chlorotrifluoroethylene copolymer (ECTFE), ethylene / trifluoroethylene copolymer, ethylene / tetrafluoroethylene copolymer (ETFE), tetrafluoroethylene / propylene copolymer (TFE / P), tetrafluoroethylene / hexafluoropropylene copolymer (FEP / HFP), hexafluoropropylene / tetrafluoroethylene / vinylidene copolymer (THV), or a perfluoro(1-butenyl vinyl ether) homocyclic polymer with functionalized polymer end groups.

[0032] In another embodiment, the elastomeric layer, such as a silicone or PMP layer, and the fluoropolymer layer of the oxygen permeable container are bonded.

[0033] In another embodiment, bonding of the oxygen permeable container is performed by coating, RF welding, ultrasonic welding, hot bar welding, chemical bonding, adhesive bonding, hot melt bonding, solvent welding, laser welding, corona discharge, radiation, lamination (e.g., by extreme heat, tape or melt lamination), etching, plasma treatment, wetting, adhesives, radiation, extrusion, coextrusion, or a combination thereof.

[0034] In another embodiment, the oxygen permeable container comprises a bag, flask, or tube.

[0035] In another embodiment, the oxygen permeable bag, flask or tube contains a sealable access port.

[0036] The present invention provides a method for culturing cells in an oxygen permeable container. The method comprises the steps of: a) adding a culture medium to the container, and b) adding cells to the container, wherein the container comprises an outer elastomeric layer, an inner fluid contact layer having a low TOC. In one aspect, the inner fluid contact layer is a fluoropolymer layer, wherein the inner fluoropolymer layer is coated onto the outer elastomeric layer, and wherein the cells and the culture medium are in contact with the inner fluoropolymer layer.

[0037] In another embodiment of the method, the outer elastomeric layer of the container is natural polyisoprene rubber (NR), synthetic polyisoprene rubber (IR), polybutadiene rubber (BR), chloroprene rubber (CR), butyl rubber (IIR), halogenated butyl rubber (CIIR, BIIR), styrene butadiene rubber (SBR), nitrile rubber (NBR) and hydrogenated nitrile rubber (HNBR), ethylene propylene rubber (EPM), ethylene propylene diene monomer rubber (EPDM), epichlorohydrin rubber (ECO), polyacrylate rubber (ACM, ABR), silicone rubber (SI, Q, VMQ), fluorosilicone rubber (FSR, FVMQ), fluoroelastomer (FKM, FEPM), perfluoroelastomer (FFKM), polyether block amide (PEBA), chlorosulfonated polyethylene (CSM), ethylene vinyl acetate (EVA), cyclic olefin copolymer, polyolefin elastomer, elastomer PET or a combination thereof.

[0038] In another embodiment of the method, the outer elastomeric layer of the container is a polymethylpentene polymer (PMP).

[0039] In another embodiment of the method, the outer elastomeric layer of the container is silicone rubber.

[0040] In another embodiment of the method, the silicone rubber of the oxygen permeable container is high consistency rubber (HCR), fluorosilicone rubber (FSR), liquid silicone rubber (LSR), or room temperature vulcanized rubber (RTV), thermoplastic silicone rubber (TPE), platinum-cured silicone rubber or peroxide-cured silicone rubber.

[0041] In yet another embodiment of the method, the silicone rubber of the oxygen permeable container is thermoset, cured, vulcanized, catalyzed, or injection molded.

[0042] In another embodiment of the method, the fluoropolymer layer of the container is fluorinated ethylene-propylene (FEP), polytetrafluoroethylene (PTFE), 3M TM Dyneon TM TFM TM Modified PTFE, polyvinylidene fluoride (PVDF), tetrafluoroethylene-perfluoro(propyl vinyl ether) (PFA), polyvinylidene fluoride (PVF), polychlorotrifluoroethylene (PCTFE), tetrafluoroethylene / hexafluoropropylene / ethylene copolymer (HTE), chlorotrifluoroethylene / vinylidene fluoride copolymer, chlorotrifluoroethylene / hexafluoropropylene, ethylene / chlorotrifluoroethylene copolymer (ECTFE), ethylene / trifluoroethylene copolymer, ethylene / tetrafluoroethylene copolymer (ETFE), tetrafluoroethylene / propylene copolymer (TFE / P), tetrafluoroethylene / hexafluoropropylene copolymer (FEP / HFP), hexafluoropropylene / tetrafluoroethylene / vinylidene copolymer (THV), or perfluoro(1-butenyl vinyl ether) homocyclic polymer with functionalized polymer end groups.

[0043] In another embodiment of the method, the elastomeric, such as PMP or silicone, layer and the fluoropolymer layer of the oxygen permeable container are bonded.

[0044] In another embodiment of the method, bonding of the oxygen permeable container is performed by chemical bonding, adhesive bonding, hot melt bonding, solvent bonding, laser welding, surface treatment, extrusion, coextrusion, coating, lamination, tape lamination, coating, or a combination thereof.

[0045] In another embodiment of the method, the oxygen permeable container comprises a bag, a flask, or a tube.

[0046] In another embodiment of the method, the oxygen permeable bag, flask or tube comprises a sealable access port.

[0047] In another embodiment of the method of culturing cells, steps a) adding culture medium to the container and b) adding cells to the container are interchangeable.

[0048] In another embodiment of the method of culturing cells, steps a) and b) are followed by incubation of the container.

[0049] Although multiple embodiments are disclosed, other embodiments of the present invention will become apparent to those skilled in the art from the detailed description below. As will be apparent, the present invention can be modified in various obvious aspects without departing from the spirit and scope of the present invention. Accordingly, the detailed description is to be regarded as illustrative rather than restrictive. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 A side view of a fluoropolymer coated elastomeric film is shown.

[0051] Figure 2 A fluoropolymer coated elastomeric culture vessel is shown.

[0052] Figure 3 Diagram showing initial ingress of oxygen into an existing FEP bag.

[0053] Figure 4 A graphical representation of the initial ingress of oxygen in a prototype bag with a gas permeable laminate is shown.

[0054] Figure 5 A graphic representation of the equilibration method in a prototype bag made with a gas permeable laminate is shown. DETAILED DESCRIPTION

[0055] In the specification and claims, the terms "comprising" and "including" are open-ended terms, and should be interpreted to mean "including, but not limited to... ." These terms encompass the more rigorous terms "consisting essentially of and "consisting of.

[0056] It must be noted that, as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural references unless the context clearly indicates otherwise. Likewise, the terms "a" (or "a"), "one or more", and "at least one" are used interchangeably herein. It should also be noted that the terms "comprising", "including", "characterized by", and "having" are used interchangeably.

[0057] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those of ordinary skill in the art to which the present invention belongs. All publications and patents specifically mentioned herein are incorporated herein by reference in their entirety for all purposes, including chemicals, apparatus, statistical analysis and methods reported in descriptions and open publications that can be used in conjunction with the present invention. All references cited in this specification are considered to be indications of the state of the art in the art. Any content herein should not be construed as admitting that the present invention has no right to be earlier than such disclosure due to prior invention.

[0058] A combination of beneficial properties of fluoropolymers and elastomeric films for application in cell culture bags is desired in the art and is provided herein.

[0059] The gas permeable membrane of the first aspect described herein comprises an elastomeric membrane coated with a fluoropolymer. Oxygen is necessary for cell growth in the culture medium, and carbon dioxide is necessary for the culture medium to buffer pH. Most normal mammalian cell lines grow well at pH 7.4, and there is minimal variability in different cell strains. However, some transformed cell lines have been shown to grow better in a slightly more acidic environment (pH 7.0–7.4), and some normal fibroblast cell lines prefer a slightly more alkaline environment (pH 7.4–7.7). Because the pH of the culture medium depends on the delicate balance of dissolved carbon dioxide and bicarbonate, changes in carbon dioxide concentration can change the pH of the culture medium. Therefore, it is necessary for the elastomeric membrane coated with a fluoropolymer used in a cell culture bag to have good gas permeability for oxygen and carbon dioxide.

[0060] Those skilled in the art will recognize that the gas permeable material should be selected based on a variety of properties, including flexibility, sealability to ensure airtightness, good transparency to allow microscopic examination of cell growth, absence of plasticizers that may be harmful to cells (e.g., dioctyl phthalate and diisodecyl phthalate), moisture vapor permeability, ability to be modified for desired cell-to-cell interactions, optical clarity, physical strength, and the like.

[0061] Elastomers are polymers that have viscoelastic properties and very weak intermolecular forces, generally with a low Young's modulus and high strain to failure compared to other materials. The term elastomer is used interchangeably with the term rubber, although rubber is preferred when referring to vulcanized rubber. Elastomers are amorphous polymers composed of monomers of carbon, hydrogen, oxygen and / or silicon.

[0062] Elastomers include unsaturated rubbers curable by sulfur vulcanization, such as natural (NR) and synthetic polyisoprene (IR), polybutadiene (BR), chloroprene rubber (CR), butyl rubber (IIR), halogenated butyl rubber (CIIR, BIIR), styrene butadiene rubber (SBR), nitrile rubber (NBR) and hydrogenated nitrile rubber (HNBR).

[0063] Elastomers include unsaturated rubbers that cannot be cured by sulfur vulcanization, such as ethylene propylene rubber (EPM), ethylene propylene diene monomer rubber (EPDM), epichlorohydrin rubber (ECO), polyacrylate rubber (ACM, ABR), silicone rubber (SI, Q, VMQ), fluorosilicone rubber (FSR, FVMQ), fluoroelastomers (FKM, FEPM), perfluoroelastomers (FFKM), polyether block amides (PEBA), chlorosulfonated polyethylene (CSM), thermoplastic polyurethanes (TPU) (including thermoplastic silicones such as ), cyclic olefin copolymers, polyolefin elastomers, elastomer PET and ethylene vinyl acetate (EVA).

[0064] Thermoplastic polyurethanes (TPUs) are known in the art. Typically, thermoplastic polyurethanes are formed by reacting a polyol with an isocyanate. The overall properties of the polyurethane will depend on the type of polyol and isocyanate, the degree of crystallinity in the polyurethane, the molecular weight of the polyurethane, and the chemical structure of the polyurethane backbone.

[0065] Depending on the degree of crosslinking present, polyurethanes can be thermoplastic or thermosetting. Depending on the functionality of the reactants, thermoplastic polyurethanes (TPUs) have no major crosslinking, while thermosetting polyurethanes have varying degrees of crosslinking.

[0066] Thermoplastic polyurethanes are usually based on methylene diisocyanate (MDI) or toluene diisocyanate (TDI), and include both polyester and polyether grade polyols. Thermoplastic polyurethanes can be formed by a "one-shot" reaction between isocyanates and polyols or by a "prepolymer" system, in which a curing agent is added to a partially reacted polyol isocyanate compound to complete the polyurethane reaction. Examples of some common thermoplastic polyurethane elastomers based on "prepolymers" are "TEXIN", a trade name of Bayer MaterialsScience, "ESTANE", a trade name of Lubrizol, "PELLETHANE", a trade name of Dow Chemical Co., and "ELASTOLLAN", a trade name of BASF, Inc.

[0067] Thermoplastic silicones include but are not limited to 140 Silicone TPE, 200 Silicone TPE elastomer (90% polydimethylsiloxane and isocyanate), 60 Silicone TPE, 80 Silicone TPE and 145TPE, these all contain 90% polydimethylsiloxane and isocyanate.

[0068] Polymethylpentene (PMP) is a thermoplastic polymer of methylpentene monomer units, and is highly gas permeable and transparent.

[0069] Silicone rubber has proven to be a particularly good material for gas permeable membranes. In order to ensure adequate oxygen and carbon dioxide exchange, the thinnest possible gas exchange membranes are preferred. Membranes with a thickness between 0.1 mm and 1 mm have proven successful.

[0070] Silicone membranes can be economically manufactured by injection molding in any desired shape. Silicone is commercially available in a variety of thicknesses, shapes and specific gas permeabilities. It has high tear resistance and good chemical resistance to the culture media commonly used in cell culture and is therefore also particularly easy to handle.

[0071] The ability to sterilize a gas permeable silicone membrane is also particularly advantageous. In particular, it can be effectively sterilized in an autoclave without substantial changes in shape and can be reused several times.

[0072] The silicone rubber used preferably has a leachable and extractable profile that is as low as possible.

[0073] In one embodiment, the cell culture vessel may comprise a single layer of an elastomer, such as a thermoplastic silicone (e.g., silicone elastomer available from Wacker Chemie AG). Polydimethylsiloxane / urea copolymer) or a fluoropolymer having TOC and permeability as described herein.

[0074] It should also be noted that other configurations of thermoplastics (elastomers and non-elastomers) and fluoropolymer configurations can also be used to control the gas permeability of composites while containing a low TOC fluid contact layer. Control of gas permeability can be used for the purpose of producing high or low gas permeability composites. Examples of thermoplastic elastomers (TPEs) include styrene block copolymers (TPE-s), olefins (TPE-o), alloys (TPE-v or TPV), polyurethanes (TPU), copolyesters and polyamides. Examples of non-elastomeric thermoplastics include acrylic acid, acrylonitrile butadiene styrene (ABS), nylon, polylactic acid (PLA), polybenzimidazole (PBI), polycarbonate (PC), polyether sulfone (PES), polyether ether ketone (PEEK), polyetherimide (PEI), polyethylene (PE), polyphenylene oxide (PPO), polyphenylene sulfide (PPS), polypropylene (PP), polystyrene (PS) and polyvinyl chloride (PVC), ethylene vinyl alcohol (EVOH) and any traditional rigid polymers whose monomer structures have been improved to reduce crystallinity and increase flexibility.

[0075] Microporous, hydrophobic fluoropolymers such as 3M TM Dyneon TM TFM TM Modified PTFE, HTE or THV, have also proven to be advantageous as materials for gas exchange membranes. The hydrophobicity of the fluoropolymers ensures that the gas exchange membranes are impermeable to the aqueous medium. For a given gas permeability, the required geometry of the gas exchange membrane depends on the gas demand caused by cellular respiration and the partial pressures of the gases involved in cellular respiration, especially the oxygen partial pressure acting on them from the outside.

[0076] Figure 1 A side view of a fluoropolymer coated elastomeric film 1 is shown. It should be understood that the fluoropolymer layer 10 may be disposed on an elastomeric layer 20 to form the film 1. The fluoropolymer layer 10 may be from a castable solution. Alternatively, the elastomer may also be cast / disposed on the fluoropolymer layer.

[0077] Typically, the fluoropolymer layer 10 has a thickness of about 0.001 mm to about 0.7 mm, more particularly about 0.005 mm to about 0.4 mm, and most particularly about 0.01 mm to about 0.1 mm, and all thicknesses therebetween, including 0.02 mm, 0.03 mm, 0.04 mm, 0.05 mm, 0.06 mm, 0.07 mm, 0.08 mm, and 0.09 mm. In a particular aspect, the fluoropolymer is a FEP layer, and the thickness of the fluoropolymer layer is 0.0254 mm or 1 mil.

[0078] The elastomer layer 20 generally has a thickness of about 0.01 mm to about 5 mm, more particularly about 0.05 mm to about 1 mm, and most particularly about 0.1 mm to about 0.5 mm, and all thicknesses therebetween, including 0.15 mm, 0.2 mm, 0.25 mm, 0.3 mm, 0.35 mm, 0.4 mm, and 0.45 mm. In a specific aspect, the elastomer is a thermosetting silicone layer, and the thickness of the elastomer layer is 0.127 mm to 0.254 mm or 5 to 10 mils.

[0079] The term "castable" is intended to mean a fluoropolymer or non-fluoropolymer that can be dispersed, dissolved, suspended, emulsified or otherwise distributed in a liquid carrier medium. The liquid carrier medium can be water, an organic solvent or any other liquid in which the polymer can be dispersed, dissolved, suspended, emulsified or otherwise distributed. The liquid carrier medium can be a mixture of suitable liquids. Once distributed in the carrier medium, the polymer and the medium can then be arranged or cast on a support material to form a film. The polymer can be mixed with a first carrier liquid. The mixture can include a dispersion of polymer particles in the first carrier liquid, an emulsion of polymer droplets, or a monomer or oligomer precursor of the polymer in the first carrier liquid or a polymer solution in the first carrier liquid. Making the polymer "castable" in addition / equivalent would be if it is heated to above its melting point or processed in a liquid state, then cured by UV, IR, initiator or electron beam so that it becomes solid.

[0080] The castable polymer may also be a monomeric or oligomeric precursor of a polymer dispersed in a carrier liquid.The most common castable compositions are emulsions or dispersions in an aqueous medium.

[0081] The selection of the first carrier liquid is based on the specific polymer and the form in which the material is to be introduced into the casting composition of the present invention. If a solution is desired, a solvent for the specific fluoropolymer is selected as the carrier liquid. Suitable carriers include, for example, DMAC, NMP, glycol ethers, or water, etc. If a dispersion is desired, a suitable carrier is one in which the polymer is insoluble. An aqueous solution would be a suitable carrier liquid for dispersion of the polymer particles.

[0082] Most commonly castable compositions are emulsions or dispersions in aqueous media. Surfactants can be used to prepare dispersions in an amount effective to improve the surface tension of the carrier fluid. Suitable surfactant compounds include ionic surfactants, amphoteric surfactants, cationic surfactants and nonionic surfactants.

[0083] Fluoropolymers are generally chosen as fluid contact layers due to their hydrophobicity and because they are biologically, chemically and immunologically inert.

[0084] The phrase "fluoropolymer" is known in the art and is intended to include, for example, fluorinated ethylene-propylene (FEP), polytetrafluoroethylene (PTFE), polyvinylidene fluoride (PVDF), polychlorotrifluoroethylene (PCTFE), polyvinylidene fluoride (PVF), tetrafluoroethylene / hexafluoropropylene / ethylene copolymer (HTE), chlorotrifluoroethylene / vinylidene fluoride copolymer, chlorotrifluoroethylene / hexafluoropropylene, ethylene / chlorotrifluoroethylene copolymer (ECTFE), ethylene / trifluoroethylene copolymer, ethylene / tetrafluoroethylene copolymer (ETFE), tetrafluoroethylene / propylene copolymer (TFE / P), tetrafluoroethylene / hexafluoropropylene copolymer (FEP / HFP), tetrafluoroethylene-perfluoro(alkyl vinyl ether) copolymer (e.g., PFA; tetrafluoroethylene-perfluoro(propyl vinyl ether), polyvinylidene fluoride, hexafluoropropylene / tetrafluoroethylene / vinylidene copolymer (THV), and mixtures thereof.

[0085] The phrase "fluoropolymer" also includes amorphous polymers that are non-crystalline or have a heat of fusion of less than 2 J / g when measured by DSC. These include copolymers of TFE with functional or non-functional monomers such as fluoroolefins having 2-8 carbon atoms and fluoroalkyl vinyl ethers in which the alkyl group contains 1 or 3 to 5 carbon atoms. Examples of non-functional monomers include hexafluoropropylene (HFP), chlorotrifluoroethylene (CTFE), PEVE, PMVE, and perfluoro-(propylene vinyl ether) (PPVE). Functional monomers include perfluoroethyl vinyl ether (EVE), CF2CFOCF2CFCF3OCF2CF2COOCH3 (EVE-carbamate), CF2CFOCF2CFCF3OCF2CF2SO2F (PSEPVE), CF2CFOCF2CFCF3OCF2CF2CN (8CNVE), N3(CF2CFOCF2CFCF3OCF2CF2)3 (EVE-triazine), CF2CFOCF2CFCF3OCF2CF2CN (EVE-CN), CF2CFOCF2CFCF3OCF2CF2 CH2OH (EVE-OH), CF2CFOCF2CFCF3OCF2CF2CH2PO2(OH)2 (EVE-P)CF2CFOCF2CFCF3OCF2CF2CH2COOH (EVE-COOH) and 2,2-bistrifluoromethyl-4,5-difluoro-1,3-dioxole (PDD).

[0086] Commercially available amorphous fluoropolymer materials include those from DuPont, Wilmington, DE: SF60(TFE / PMVE / PEVE,DuPont,Wilmington DE)、 SF61(TFE / PMVE / PEVE / EVE-P), SF50(TFE / HFP), AF 1600 (PDD / TFE) and AF2130 (PDD / CTFE); Asahi Corporation, Tokyo, Japan CYTOP A, CYTOP M, CYTOP S, or CYTOP NM); from MY Polymers Corporation in Rehovot, Israel (MY-133); or from Nusil Corporation in Carpinteria, CA (LS-233).

[0087] The fluoropolymer solution may be applied by conventional coating methods including, but not limited to, spraying, dipping, brushing, or extrusion / injection molding.

[0088] It is a perfluoro(1-butenyl vinyl ether) homocyclic polymer containing functionalized end groups. It exists as an amorphous resin and is available in various concentrations in perfluorinated solvents and select non-fluorinated solvent systems. For example: 0.1 to 1% in 9:5 isopropanol / isobutyl acetate.

[0089] CYTOP A type contains carboxyl polymer end groups and CYTOP M type contains amino-silane polymer end groups. Either fluoropolymer can be used with accelerators, special primers or as a silane coupling agent for plastic coating applications. CYTOP S type contains all-fluoropolymer end groups for non-stick UV resistance and high transparency.

[0090] Silicone rubber formulations include silicone polymer matrices. The polymer matrix can be formed, for example, using non-polar silicone polymers. Silicone polymers can, for example, include polyalkylsiloxanes, such as silicone polymers formed from precursors, such as dimethylsiloxane, diethylsiloxane, dipropylsiloxane, methylethylsiloxane, methylpropylsiloxane or a combination thereof. In particular, polyalkylsiloxanes include polydialkylsiloxanes, such as polydimethylsiloxane (PDMS). In general, silicone polymers are non-polar and do not contain halogen functional groups, such as chlorine and fluorine, and do not contain phenyl functional groups.

[0091] Functionalized polyorganosiloxanes include dimethylsiloxane terminated at both ends by vinyldimethylsiloxy groups, dimethylsiloxane-vinylmethylsiloxane copolymer terminated at both ends by vinyldimethylsiloxy groups, and dimethylsiloxane-methylphenylsiloxane copolymer terminated at both ends by vinyldimethylsiloxy groups.

[0092] The silicone rubber may comprise a platinum-catalyzed liquid silicone rubber (LSR) or a high consistency gum rubber (HCR). The silicone rubber may also comprise a peroxide-catalyzed silicone rubber (LSR) or a high consistency gum rubber (HCR). For example, Saint-Gobain produces a silicone rubber made of (peroxide-based silicone). An example of a silicone rubber that is an HCR is GE 94506 HCR available from GE Plastics. Examples of LSRs include Wacker 3003 available through Wacker Silicone of Adrian, MI and Rhodia 4360 available through Rhodia Silicones of Ventura, CA.

[0093] In one aspect of the present invention, when silicone rubber is used as the base rubber, the composition of the fluorine-containing polymer coating may contain a reactive compound that binds to the silicone rubber. Examples of the reactive compound that binds to the silicone rubber include particularly preferred hydrocarbon-containing silicon compounds.

[0094] Examples of such silicon-containing compounds include compounds having a siloxane bond, silane coupling agents having an alkoxysilyl group, functional silanes or silazanes having a chlorosilyl group, aminosilanes, and silylating agents. Among them, compounds having a siloxane bond and silane coupling agents having an alkoxysilyl group are preferred. Examples of such compounds having a siloxane bond include terminal-modified dimethylsiloxane, condensation-type or addition-type liquid silicones, silicates, and acrylic silicone polymers.

[0095] An example of a fluoropolymer with amino-silane terminal modification is CYTOP M type. Similarly, an example of a fluoropolymer with carboxyl terminal modification is CYTOP A type. Either may be mixed with or without a coupling agent and bonded directly or indirectly to the silicone rubber via a coupling agent. Chemical bonding may occur by condensation, alkylation, amidation, silanization, etherification or via a coupling agent by any bonding combination thereof. For example, a coupling agent that may be used with silicone rubber is an isocyanate compound that reacts with hydroxyl, amino or thiol groups. After the chemical bond is formed between the fluoropolymer and the silicone rubber, adhesion will be achieved and the composite gas permeable membrane will have higher strength with minimal leaching. CYTOP A type or CYTOP M type after hydrolysis may also adhere to the silicone rubber by hydrogen bonding.

[0096] The bonding of the elastomer layer and the fluoropolymer layer together can also be achieved by laser welding, surface treatment, radiation, extrusion, coextrusion, coating, lamination, wetting, adhesives or a combination thereof. Preferred methods of surface treatment include C treatment (Saint-Gobain Performance Plastics Corporation, U.S. Patent No. 6,726,979), corona discharge, plasma treatment, etching or a combination thereof. Surface treatment can also involve chemical treatment with additives or primers that can be used alone or in combination with other disclosed treatment methods.

[0097] In a preferred aspect, the fluoropolymer layer is C-treated FEP which has also been pre-treated and coated with LSR silicone by extrusion.

[0098] These layers can also be similar to The laminates were pre-laminated together with that described by the Complete product (Saint-Gobain Performance Plastics Corporation, Valley Forge, PA).

[0099] Total organic carbon (TOC) is the amount of carbon bound in an organic compound and is often used as a nonspecific indicator for pharmaceutical manufacturing equipment, among other things. TOC is used as a process control attribute in the biotechnology industry to monitor the performance of unit operations employing purification and distribution systems.

[0100] In a specific embodiment, TOC can be measured according to US Pharmacopeia (USP) 643 and an apparatus that utilizes a high temperature wet oxidation reaction (UV-promoted chemical oxidation) Ultra-Clean Technology Handbook: Volume 1: Ultra-PureWater , Ohmi, Tadahiro; CRC Press, 1993, pp. 497-517). Purified water is placed in contact with the polymer at 70°C for 24 hours, for example, at 3 cm 2 The ratio of the surface area of ​​the article to 1 mL of water. The water is removed from contact with the polymer and tested in a TOC analyzer. A suitable device is a TEKMAR DOHRMANN model Phoenix 8000 TOC analyzer.

[0101] In certain embodiments, TOC can be measured for containers employed in the systems of the present disclosure, including, for example, by extracting the TOC from the inner surface area of ​​the container (wherein the TOC is expressed as mg / cm 2The results are for TOC / square centimeter of internal area). In a specific embodiment, and by way of example only, the container may be extracted in purified water at 70±2°C for 24±2 hours. The extract may be analyzed for TOC by converting the TOC to carbon dioxide, for example, by acidification with sodium persulfate and chemical wet oxidation. The carbon dioxide released from the container may be measured using an infrared detector. An example of an extraction rate for a FEP container is 3 cm 2 / mL (typical extraction rate). For some containers (such as FEP bags), no dilution is required because the TOC level is below the upper limit of the calibration curve, while for other embodiments (such as silicone tubing), dilution is required because the level of TOC is detected in the extract.

[0102] An example of TOC in an FEP container is 0.145 mg / L (0.00005 mg / cm 2 or 0.001 mg / g). For the embodiment using silicone tubing, the extraction ratio may be 14.6 cm 2 / mL (e.g. for Biosil), or 15.9 cm 2 / mL (e.g. for SR139), and an example of TOC for silicone Biosil tube is 302 mg / L (0.021 mg / cm 2 or 0.023 mg / cm), and the example of TOC of silicone SR139 pipe is 120 mg / L (0.008 mg / cm 2 or 0.0009 mg / cm). In at least some silicone tubing embodiments, samples may be diluted because the volume and concentration of the extracts result in values ​​above the maximum detection value of the machine. The dilution and different extraction ratios require that comparisons of these samples to the bag samples be made on a weight / area basis instead.

[0103] Those skilled in the art recognize that TOC values ​​can be expressed in terms of weight / volume. However, those skilled in the art know that the ratio of containers (particularly FEP bag materials) is distinguishable relative to the ratio of silicone tubing; silicone tubing values ​​can only be expressed in mg / cm 2 The weight / area result can be considered as a starting point, as this value is independent of the extraction ratio / dilution. A person skilled in the art can use the weight / area result as a basis and assume a standard 3 cm 2 / mL extraction ratio (as an example) to calculate a "normalized" weight / volume ratio in order to compare weight / volume values.

[0104] In a specific embodiment, the TOC of the thermoplastic elastomer (TPE) is 0.002 mg / cm 2 (0.032 mg / g or 5.88 mg / mL). In certain embodiments, the TOC of the FEP is 0.00005 mg / cm of the interior wetted surface of the article. 2(0.001 mg / g or 0.145 mg / mL of the article). In a specific embodiment, the TOC of the silicone is 0.021 mg / cm on the interior wetted surface of the article. 2 Or 63mg / mL. In a specific embodiment, the TOC of the PMP membrane is 0.07ppm (0.00001mg / cm 2 ).

[0105] In a specific embodiment, the TOC of fluorinated ethylene propylene (FEP) is 0.00005 mg / cm 2 (0.001mg / g); the TOC of silicone materials such as silicone pipes is 0.021mg / cm 2 (0.023mg / cm2) and 0.008mg / cm2 2 (0.009 mg / cm); the TOC of cell culture bags used historically was 0.002 mg / cm 2 (0.032mg / g).

[0106] Those skilled in the art will recognize that if mg / cm 2 If the unit is mg / L, the extraction ratio must be known. The conversion can occur as follows: the machine outputs a value in mg / L, the dilution is taken into account, and then the surface area and total volume are used to convert that number to mg / cm 2 An example of Silicone Biosil is provided: Silicone Biosil sample: 302mg / L*1L / 1000mL*23.7mL / 347cm 2 =.021mg / cm 2 .

[0107] In certain embodiments, the 2 Units compare TOC because no extraction ratio or any dilution is required.

[0108] Below is an example of TOC calculation for a Silicone Tubing Biosil sample and a Silicone Tubing SR139 sample.

[0109] Test article extraction

[0110] sample <![CDATA[Inner surface area (cm 2 )]]> Length (cm) Volume of pure water (mL) Silicone Tube Biosil Sample 347 314 23.7 Silicone tube SR139 sample 342 295 21.5

[0111] TOC Analysis Results

[0112]

[0113] Below is an example of TOC calculation for a FEP bag and a monolayer bag that consists primarily of SEBS (styrene block copolymer) and may also contain EVA and PP):

[0114] Test article extraction

[0115] sample <![CDATA[Inner surface area (cm 2 )]]> Weight(g) Volume of pure water (mL) FEP bag 650 30.9 217 SEBS bag 362.9 22.5 121

[0116] TOC Analysis Results

[0117] sample mg / L <![CDATA[mg / cm 2 ]]> mg / g Detection limit (mg / L) FEP bag 0.145 0.00005 0.001 0.1 SEBS bag 5.88 0.002 0.032 0.1

[0118] In a specific embodiment, the container comprises an inner surface comprising a 2 , 0.9mg / cm2, 0.8mg / cm 2 , 0.7mg / cm 2 , 0.6mg / cm 2 , 0.5mg / cm 2 , 0.4mg / cm 2 , 0.3mg / cm 2 , 0.2mg / cm 2 , 0.1mg / cm 2 , 0.09mg / cm 2 , 0.08mg / cm 2 , 0.07mg / cm 2 , 0.06mg / cm 2 , 0.05mg / cm 2 , 0.04mg / cm 2 , 0.03mg / cm 2 , 0.02mg / cm 2 , 0.01mg / cm 2 , 0.009mg / cm 2 , 0.008mg / cm 2 , 0.007mg / cm 2 , 0.006mg / cm 2 , 0.005mg / cm 2 , 0.004mg / cm 2 , 0.003mg / cm 2 , 0.002mg / cm 2 , 0.001mg / cm 2 Equivalent (or undetectable) total organic carbon (TOC) aggregates in water.

[0119] In certain embodiments, the TOC in water is less than 0.001 mg / cm 2 Up to 0.1mg / cm 2 , 0.001mg / cm 2 Up to 0.095mg / cm 2 , 0.001mg / cm2 Up to 0.075mg / cm 2 , 0.001mg / cm 2 To 0.05mg / cm2, 0.001mg / cm 2 To 0.01mg / cm2, 0.001mg / cm 2 To 0.005mg / cm 2 , or 0.001mg / cm 2 Up to 0.025mg / cm 2 In certain embodiments, the TOC in water is less than 0.01 mg / cm 2 Up to 0.1mg / cm 2 , 0.01mg / cm 2 Up to 0.075mg / cm 2 , 0.01mg / cm 2 Up to 0.05mg / cm 2 , or 0.01mg / cm 2 Up to 0.025mg / cm 2 In certain embodiments, the TOC in water is less than 0.05 mg / cm 2 Up to 0.1mg / cm 2 , 0.05mg / cm 2 Up to 0.09mg / cm 2 , 0.05mg / cm 2 Up to 0.075mg / cm 2 , or 0.05mg / cm 2 Up to 0.06mg / cm 2 In certain embodiments, the TOC in water is less than 0.005 mg / cm 2 Up to 0.1mg / cm 2 , 0.005mg / cm 2 Up to 0.095mg / cm 2 , 0.005mg / cm 2 Up to 0.075mg / cm 2 , 0.005mg / cm 2 Up to 0.05mg / cm 2 , 0.005mg / cm 2 Up to 0.025mg / cm 2 , or 0.005mg / cm 2 To 0.01mg / cm 2 Amount within range.

[0120] The material used for the oxygen permeable membrane of the present embodiment may have a determined permeability. The permeability of the oxygen permeable elastomeric membrane is at least 100 cc / m 2 / day, preferably at least 500cc / m 2 / day, preferably at least 1000cc / m 2 / day, preferably at least 1500cc / m 2 / day, and most preferably at least 2000cc / m 2 / day, and even more preferably at least 2200cc / m 2 / day. The oxygen permeability of the elastomeric fluoropolymer composite membrane is at least 100 cc / m 2 / day, preferably at least 500cc / m 2 / day, preferably at least 1000cc / m 2 / day, preferably at least 1500cc / m 2 / day, and most preferably at least 2000cc / m 2 / day, and even more preferably at least 2200cc / m 2 / day. Oxygen permeability was measured at 25°C using a MOCON Ox-tran 2 / 21H oxygen analyzer following ASTM D3985. In another aspect of membrane permeability, the standardized units (cc-mm / m 2 -day) can be used to display any thickness of the film. For example, at a temperature of 25 ° C, the conversion range of 5 mil film is about 12.7 cc-mm / m 2 -Daily to at least about 279cc-mm / m 2 -days. The permeability of the structure / composite material can be maintained at cc / m 2 of scope, as it will consist of two layers.

[0121] Figure 2 is a view of a fluoropolymer coated elastomeric cell culture container 25. A fluoropolymer layer 10 (not shown) comprises an inner layer of a membrane 1 that contacts the cell culture. The fluoropolymer coated elastomeric cell culture container 25 comprises at least one fluorinated elastomeric membrane 1 bonded to form a cell culture compartment 30, which is also bonded to at least one sealable access port 40 that allows access to the interior of the container. The fluoropolymer coated elastomeric cell culture container 25 comprises a cell culture compartment 30, which can be a bag, flask or tube comprising a closed system, which also comprises at least one sealable access port 40.

[0122] The bonding of the membrane 1 to form the cell culture container 30 and the bonding of the access port 40 to form the cell culture container 30 are each independently accomplished by: RF welding, ultrasonic welding, hot rod welding, chemical bonding, adhesive bonding, hot melt bonding, solvent welding, laser welding, corona discharge, radiation, for example, by extremely hot lamination or melt lamination, etching, plasma treatment, wetting, adhesive, radiation, extrusion, coextrusion or a combination thereof. Preferred methods of surface treatment include C treatment (Saint-Gobain Performance Plastics Corporation, U.S. Pat. No. 6,726,979), corona discharge, plasma treatment, etching or a combination thereof. Surface treatment may also involve chemical treatment with additives or primers that may be used alone or in combination with other disclosed treatment methods. The bonding of the access port 40 may occur before, after or during the formation of the cell culture container 30.

[0123] The cell culture container 30 includes at least one access port 40 that allows access to the interior of the container. The access port 40 may include a sealable cap or screw cap, a pierceable material or septum, or a valve assembly that allows access through a tube or syringe. A disposable sensor that measures pH / DO may also be included in the container to monitor cell culture conditions.

[0124] The location of the access port is not limited to the current configuration, and the port may be positioned for ease of use or manufacturing. The access port comprises a fluoropolymer tube attached to the composite container by RF welding, ultrasonic welding, hot bar welding, chemical bonding, adhesive bonding, heat melt bonding, solvent welding, laser welding, corona discharge, radiation, lamination such as by extreme heat or melt lamination, etching, plasma treatment, wetting, adhesives, radiation, coextrusion, or a combination thereof.

[0125] The volume of the cell culture container 30 may range from 1 mL to 50 L, 50 mL to 10 L, but is preferably 50 mL to 1 L.

[0126] Suitable growth media include, for example, nutrients or lysogeny with added hormones and / or growth factors.

[0127] Suitable cells include, for example, connective tissue cells, bone cells, cardiac cells, epithelial cells, neural cells, endocrine cells, immune cells, lymphocytes, melanocytes, tumor cells, or mixtures thereof.

[0128] In one embodiment, in the first paragraph (1), the present invention provides an oxygen permeable membrane comprising a first elastomeric layer; having a molecular weight of less than 1 mg / cm 2 A second fluid contacting layer of total organic carbon (TOC) in water.

[0129] 2. A membrane according to paragraph 1 wherein the second fluid contacting layer is a fluoropolymer layer, and wherein the first elastomeric layer and the second fluoropolymer layer are adhered together.

[0130] 3. A film according to paragraph 1 or 2, wherein the first elastomeric layer comprises natural polyisoprene rubber (NR), synthetic polyisoprene rubber (IR), polybutadiene rubber (BR), chloroprene rubber (CR), butyl rubber (IIR), halogenated butyl rubber (CIIR, BIIR), styrene butadiene rubber (SBR), nitrile rubber (NBR) and hydrogenated nitrile rubber (HNBR), ethylene propylene rubber (EPM), ethylene propylene diene monomer rubber (EPDM), epichlorohydrin rubber (ECO), polyacrylate rubber (ACM, ABR), silicone rubber (SI, Q, VMQ), fluorosilicone rubber (FSR, FVMQ), fluoroelastomer (FKM, FEPM), perfluoroelastomer (FFKM), polyether block amide (PEBA), chlorosulfonated polyethylene (CSM), ethylene vinyl acetate (EVA), cyclic olefin copolymer, polyolefin elastomer, elastomer PET or a mixture thereof.

[0131] 4. A film according to paragraph 1 or 2 wherein the first elastomeric layer is a polymethylpentene polymer (PMP).

[0132] 5. A film according to paragraph 1 or 2 wherein the first elastomeric layer is silicone rubber.

[0133] 6. A membrane according to paragraph 5, wherein the silicone rubber is a high consistency rubber (HCR), a fluorosilicone rubber (FSR), a liquid silicone rubber (LSR), a room temperature vulcanized rubber (RTV), a thermoplastic silicone rubber (TPE), a platinum-cured silicone rubber or a peroxide-cured silicone rubber.

[0134] 7. A membrane according to paragraph 6 wherein the silicone rubber is thermoset, cured, vulcanized, catalyzed or injection molded.

[0135] 8. A membrane according to any one of paragraphs 2 to 7, wherein the second fluoropolymer layer is fluorinated ethylene-propylene (FEP), polytetrafluoroethylene (PTFE), 3M TM Dyneon TM TFM TMModified PTFE, polyvinylidene fluoride (PVDF), tetrafluoroethylene-perfluoro(propyl vinyl ether) (PFA), polyvinylidene fluoride (PVF), polychlorotrifluoroethylene (PCTFE), tetrafluoroethylene / hexafluoropropylene / ethylene copolymer (HTE), chlorotrifluoroethylene / vinylidene fluoride copolymer, chlorotrifluoroethylene / hexafluoropropylene, ethylene / chlorotrifluoroethylene copolymer (ECTFE), ethylene / trifluoroethylene copolymer, ethylene / tetrafluoroethylene copolymer (ETFE), tetrafluoroethylene / propylene copolymer (TFE / P), tetrafluoroethylene / hexafluoropropylene copolymer (FEP / HFP), hexafluoropropylene / tetrafluoroethylene / vinylidene copolymer (THV), or perfluoro(1-butenyl vinyl ether) homocyclic polymer with functionalized polymer end groups.

[0136] 9. The film according to any of paragraphs 2 to 8, wherein the first elastomeric layer and the second fluoropolymer layer are adhered together by bonding.

[0137] 10. The film according to paragraph 9, wherein the bonding is performed by chemical bonding, adhesive bonding, hot melt bonding, solvent bonding, laser welding, surface treatment, extrusion, coextrusion, coating, lamination, or a combination thereof.

[0138] 11. An oxygen permeable container comprising an outer elastomeric layer; and having a molecular weight of less than 1 mg / cm 2 The internal fluid contact layer of the total organic carbon (TOC) in water.

[0139] 12. A container according to paragraph 11 wherein the fluid contacting layer is a fluoropolymer layer, and wherein the inner fluoropolymer layer is adhered to an outer elastomeric layer of the container.

[0140] 13. A container according to paragraph 11 or 12, wherein the outer elastomeric layer comprises natural polyisoprene rubber (NR), synthetic polyisoprene rubber (IR), polybutadiene rubber (BR), chloroprene rubber (CR), butyl rubber (IIR), halogenated butyl rubber (CIIR, BIIR), styrene butadiene rubber (SBR), nitrile rubber (NBR) and hydrogenated nitrile rubber (HNBR), ethylene propylene rubber (EPM), ethylene propylene diene monomer rubber (EPDM), epichlorohydrin rubber (ECO), polyacrylate rubber (ACM, ABR), silicone rubber (SI, Q, VMQ), fluorosilicone rubber (FSR, FVMQ), fluoroelastomer (FKM, FEPM), perfluoroelastomer (FFKM), polyether block amide (PEBA), chlorosulfonated polyethylene (CSM), ethylene vinyl acetate (EVA), cyclic olefin copolymer, polyolefin elastomer, elastomeric PET or a mixture thereof.

[0141] 14. A container according to paragraph 11 or 12, wherein the outer elastomeric layer is a polymethylpentene polymer (PMP).

[0142] 15. A container according to paragraph 11 or 12, wherein the outer elastomeric layer is silicone rubber.

[0143] 16. A container according to paragraph 15, wherein the silicone rubber is high consistency rubber (HCR), fluorosilicone rubber (FSR), liquid silicone rubber (LSR), or room temperature vulcanized rubber (RTV), thermoplastic silicone rubber (TPE), platinum-cured silicone rubber or peroxide-cured silicone rubber.

[0144] 17. A container according to paragraph 16, wherein the silicone rubber is thermoset, cured, vulcanized, catalyzed or injection molded.

[0145] 18. A container according to any one of paragraphs 12 to 17, wherein the inner fluoropolymer layer is fluorinated ethylene-propylene (FEP), polytetrafluoroethylene (PTFE), 3M TM Dyneon TM TFM TM Modified PTFE, polyvinylidene fluoride (PVDF), tetrafluoroethylene-perfluoro(propyl vinyl ether) (PFA), polyvinylidene fluoride (PVF), polychlorotrifluoroethylene (PCTFE), tetrafluoroethylene / hexafluoropropylene / ethylene copolymer (HTE), chlorotrifluoroethylene / vinylidene fluoride copolymer, chlorotrifluoroethylene / hexafluoropropylene, ethylene / chlorotrifluoroethylene copolymer (ECTFE), ethylene / trifluoroethylene copolymer, ethylene / tetrafluoroethylene copolymer (ETFE), tetrafluoroethylene / propylene copolymer (TFE / P), tetrafluoroethylene / hexafluoropropylene copolymer (FEP / HFP), hexafluoropropylene / tetrafluoroethylene / vinylidene copolymer (THV), or perfluoro(1-butenyl vinyl ether) homocyclic polymer with functionalized polymer end groups.

[0146] 19. The container according to any of paragraphs 12 to 18, wherein the outer elastomeric layer and the inner fluoropolymer layer are adhered together by bonding.

[0147] 20. The container according to paragraph 19, wherein the bonding is performed by chemical bonding, adhesive bonding, hot melt bonding, solvent bonding, laser welding, and surface treatment, extrusion, coextrusion, coating, lamination or a combination thereof.

[0148] 21. The container according to any one of paragraphs 11 to 20, wherein the container comprises a bag, a flask or a tube.

[0149] 22. A container according to paragraph 21, wherein the bag, flask or tube comprises at least one sealable access port.

[0150] 23. A method of culturing cells in an oxygen permeable container comprising the steps of: a) adding a culture medium to the container; and b) adding cells, optionally together with the culture medium, to the container, wherein the container comprises an outer elastomeric layer and has an oxygen permeable capacity of less than 1 mg / cm 2 The internal fluid contact layer of the total organic carbon (TOC) in water.

[0151] 24. The method according to paragraph 23, wherein the inner fluid contacting layer is a fluoropolymer layer, and wherein the inner fluoropolymer layer is adhered to an outer elastomeric layer of the container.

[0152] 25. A method according to paragraph 23 or 24, wherein the outer elastomeric layer is natural polyisoprene rubber (NR), synthetic polyisoprene rubber (IR), polybutadiene rubber (BR), chloroprene rubber (CR), butyl rubber (IIR), halogenated butyl rubber (CIIR, BIIR), styrene butadiene rubber (SBR), nitrile rubber (NBR) and hydrogenated nitrile rubber (HNBR), ethylene propylene rubber (EPM), ethylene propylene diene monomer rubber (EPDM), epichlorohydrin rubber (ECO), polyacrylate rubber (ACM, ABR), silicone rubber (SI, Q, VMQ), fluorosilicone rubber (FSR, FVMQ), fluoroelastomer (FKM, FEPM), perfluoroelastomer (FFKM), polyether block amide (PEBA), chlorosulfonated polyethylene (CSM), cyclic olefin copolymer, polyolefin elastomer, elastomer PET and ethylene vinyl acetate (EVA).

[0153] 26. The method according to paragraph 23 or 24, wherein the outer elastomeric layer is a polymethylpentene polymer (PMP).

[0154] 27. The method according to paragraph 23 or 24, wherein the outer elastomeric layer is silicone rubber.

[0155] 28. The method according to paragraph 27, wherein the silicone rubber is a high consistency rubber (HCR), a fluorosilicone rubber (FSR), a liquid silicone rubber (LSR), or a room temperature vulcanized rubber (RTV), a thermoplastic silicone rubber (TPE), a platinum-cured silicone rubber or a peroxide-cured silicone rubber.

[0156] 29. The method according to paragraph 28, wherein the silicone rubber is thermoset, cured, vulcanized, catalyzed or injection molded.

[0157] 30. The method according to any of paragraphs 24 to 29, wherein the inner fluoropolymer layer is fluorinated ethylene-propylene (FEP), polytetrafluoroethylene (PTFE), 3M TM Dyneon TM TFMTM Modified PTFE, polyvinylidene fluoride (PVDF), tetrafluoroethylene-perfluoro(propyl vinyl ether) (PFA), polyvinylidene fluoride (PVF), polychlorotrifluoroethylene (PCTFE), tetrafluoroethylene / hexafluoropropylene / ethylene copolymer (HTE), chlorotrifluoroethylene / vinylidene fluoride copolymer, chlorotrifluoroethylene / hexafluoropropylene, ethylene / chlorotrifluoroethylene copolymer (ECTFE), ethylene / trifluoroethylene copolymer, ethylene / tetrafluoroethylene copolymer (ETFE), tetrafluoroethylene / propylene copolymer (TFE / P), tetrafluoroethylene / hexafluoropropylene copolymer (FEP / HFP), hexafluoropropylene / tetrafluoroethylene / vinylidene copolymer (THV), or perfluoro(1-butenyl vinyl ether) homocyclic polymer with functionalized polymer end groups.

[0158] 31. The method of any of paragraphs 24 to 30, wherein the outer elastomeric layer and the inner fluoropolymer layer are adhered together by bonding.

[0159] 32. The method according to paragraph 31, wherein the bonding is performed by chemical bonding, adhesive bonding, hot melt bonding, solvent bonding, laser welding, and surface treatment, extrusion, coextrusion, coating, lamination or a combination thereof.

[0160] 33. The method according to any of paragraphs 23 to 32, wherein the container comprises a bag, a flask, or a tube.

[0161] 34. The method according to paragraph 33, wherein the bag, flask or tube comprises at least one sealable access port.

[0162] 35. The method according to paragraph 23, wherein steps a) and b) are interchangeable.

[0163] 36. The method according to paragraph 23, further comprising the step of incubating the container.

[0164] Examples

[0165] The following examples are included to demonstrate preferred embodiments of the present invention. It will be appreciated by those skilled in the art that the techniques disclosed in the following examples represent techniques discovered by the inventors to work well in the practice of the present invention, and therefore can be considered to constitute preferred modes for its practice. However, in light of this disclosure, it will be appreciated by those skilled in the art that many changes may be made in the specific embodiments disclosed, and still obtain the same or similar results, without departing from the spirit and scope of the present invention.

[0166] Example 1

[0167] (Test sample)

[0168] Table 1: TOC data

[0169]

[0170] ppm = mg / L Example 2

[0171] (Dissolved oxygen experiment)

[0172] Existing FEP bags made of 5mil FEP on the top and bottom, and prototype bags made of 5mil FEP on the top and thermoplastic silicone / FEP on the bottom were filled with tap water to 300mL and 190mL, respectively. These volumes are related to a 1 cm thickness of water in the bag when the bag is lying flat, which is a well-known recommendation in the industry. A port was cut out of each bag to feed in a nitrogen line to purge the water in the bag to a level below saturation. Dissolved oxygen (DO) levels were monitored using a PreSens probe fixed to the outside of the bag and aligned with the sensor point on the inside of the bag. When the DO level in the water reached a fairly low value (~6-8%), the nitrogen line was removed from the bag and the port opening was clamped. For the remainder of the experiment, the bag and stand were returned to the horizontal static position. The PreSens probe was programmed to obtain DO measurements at specified time points to measure the ingress of oxygen as a function of time. It was assumed that the only way for oxygen to enter was through the wall of the bag.

[0173] result:

[0174] The measurements from the PreSens system were downloaded and plotted in Excel for both bags ( Figure 3 , 4 and 5). The data were studied and observations were made to group the data into categories. For example, for the prototype bag, the start measurement was excluded because the bag was moved and manipulated to remove air bubbles from above the sensor. Also, because the prototype bag was measured for a longer period of time, the data was divided into two linear sections, "initial entry" and "equilibrium method". For this reason, the best comparison with the available existing data is in Figure 3 and Figure 4 , which demonstrates the initial entry of oxygen into each bag as a function of time when starting with similar DO levels (~6-8%) for a similar amount of time (~68-70 minutes). Figure 3 and 4 , a comparison of the slopes can be made to understand the difference in ingress rate between the existing bag and the prototype bag. The existing bag has a rate of 0.008% DO / minute, while the prototype bag has a rate of 0.06% DO / minute, a difference of over 7x. This suggests the positive impact of a more permeable material on oxygen ingress.

[0175] Although the present invention has been described with reference to preferred embodiments, those skilled in the art will recognize that changes can be made in form and detail without departing from the spirit and scope of the present invention. All references cited throughout the specification, including those in the background, are incorporated herein in their entirety. Those skilled in the art will recognize, or be able to determine, many equivalents to the specific embodiments of the present invention specifically described herein using no more than routine experimentation. Such equivalents are intended to be included within the scope of the appended claims.

Claims

1. An oxygen permeable container, the oxygen permeable container comprising an oxygen permeability of at least 2000 cc / m 2 / day of at least one oxygen permeable membrane, the oxygen permeable membrane comprising: an outer elastomeric layer which is a silicone rubber layer having a thickness ranging from 0.05 mm to 1 mm; and Less than 0.1 mg / cm2 as measured according to USP 643 2 an inner fluid contact layer having a total organic carbon (TOC) in water of 0.1 to 1 mm, wherein the inner fluid contact layer is a fluorinated ethylene-propylene (FEP) layer, and wherein the inner fluid contact layer is bonded to the outer elastomeric layer of the oxygen permeable membrane by a method comprising at least corona treating the inner fluid contact layer, The container is in the form of a cell culture bag.

2. The container of claim 1, wherein the silicone rubber is high consistency rubber (HCR), fluorosilicone rubber (FSR), liquid silicone rubber (LSR), or room temperature vulcanized rubber (RTV), thermoplastic silicone rubber (TPE), platinum-cured silicone rubber, or peroxide-cured silicone rubber.

3. The container of claim 2, wherein the silicone rubber is thermoset, cured, vulcanized, catalyzed or injection molded.

4. The container of claim 1, wherein the corona treatment is a C treatment.

5. The container of claim 1, wherein the bag comprises at least one sealable access port.

6. The container of claim 11, wherein the TOC of the inner fluid contact layer in water is less than 0.01 mg / cm 2 .

7. A method for culturing cells in an oxygen permeable container according to any one of claims 1 to 6, the method comprising the following steps in any order: a) adding culture medium to the container; and b) adding cells, optionally together with said culture medium, to said container.

8. The method of claim 7, further comprising the step of incubating the container.

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