# US 6,297,046 B1 · Multilayer gas-permeable container for the culture of adherent and non-adherent cells > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 6,297,046 B1 - Document ID: US6297046B1 - Status: Granted - Kind: B1 - Application number: 08/330,717 - Filed: 1994-10-28 - Granted: 2001-10-02 - Earliest claimed: 1994-10-28 - Assignees: Baxter International Inc. - Inventors: Sidney T. Smith; David V. Bacehowski; William Kolanko; Larry Rosenbaum; Stephen L. Smith; James G. Bender; Lecon Woo; Michael T. K. Ling - Web page: https://patentgrep.com/patent/US6297046B1 ## CPC - C12M 23/14: {Bags} - C12M 23/24: {Gas permeable parts} - C12M: APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; {APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS} ## Abstract A multi-layer, flexible, gas-permeable film (10) suitable for forming a cell culture container (20), the film (10) comprising a first layer (12) composed of a polystyrene having a thickness within the range of 0.0001 inches to about 0.0010 inches and, a second layer (14) adhered to the first layer (12) composed of a polyolefin having a thickness within the range of 0.004 inches to about 0.015 inches. ## Claims ### Claim 1 A multi-layer, flexible, gas-permeable film suitable for forming a cell culture container, the film comprising: a first layer composed of a polystyrene having a thickness within the range of 0.0001 inches to about 0.0010 inches, the first layer defining an inner cell growth surface; and, a second outer layer adhered to the first layer composed of a polymer alloy blend having multiple components, the second layer having a thickness within the range of 0.004 inches to about 0.015 inches. ### Claim 2 (depends on 1) The film of claim 1 wherein the polystyrene of the first layer is modified by polybutadiene rubber. ### Claim 3 (depends on 1) The film of claim 1 wherein the first layer has a thickness in the range of about 0.0002 inches to about 0.0006 inches. ### Claim 4 (depends on 1) The film of claim 1 wherein the first layer has a thickness of about 0.0003 inches. ### Claim 5 (depends on 1) The film of claim 1 wherein the polymer alloy has three components, a first component is a styrene-ethylene-butadiene-styrene block copolymer, a second component is ethylene vinyl acetate, and a third component is polypropylene. ### Claim 6 (depends on 5) The film of claim 5 wherein the styrene-ethylene-butadiene-styrene block copolymer constitutes 40 to 85% by weight of the polymer alloy, the ethylene vinyl acetate constitutes 0 to 40% by weight of the polymer alloy, and the polypropylene constitutes 10 to 40% by weight of the polymer alloy. ### Claim 7 (depends on 1) The film of claim 1 wherein the polymer alloy has four components. ### Claim 8 (depends on 7) The film of claim 7 wherein the four component polymer alloy has a first component of a polypropylene, a second component selected from the group consisting essentially of an ultra low density polyethylene and polybutene-1, a third component of a dimer fatty acid polyamide, and a fourth component of a styrene-ethylene-butadiene-styrene block copolymer. ### Claim 9 (depends on 8) The film of claim 8 wherein the first component constitutes within the range of 30-60% by weight of the polymer alloy, the second component constitutes within the range of 25%-50% by weight of the polymer alloy, the third component constitutes within the range of 5%-40% by weight of the polymer alloy, and the fourth component constitutes 5%-40% by weight of the polymer alloy. ### Claim 10 (depends on 1) The film of claim 1 further comprising a tie layer interposed between the first and second layers, the tie layer providing adhesive compatibility between the first and second layers. ### Claim 11 (depends on 10) The film of claim 10 wherein the tie layer is composed of a gas permeable olefin. ### Claim 12 (depends on 11) The film of claim 11 wherein the gas permeable olefin is an ethylene polymer containing vinyl acetate within the range of 16%-32% by weight. ### Claim 13 (depends on 12) The film of claim 12 wherein the ethylene vinyl acetate has a vinyl acetate content of 28% by weight. ### Claim 14 (depends on 1) The film of claim 1 further comprising a skin layer adhered to the second layer opposite the first layer, the skin layer providing additional strength and scratch resistance. ### Claim 15 (depends on 14) The film of claim 14 wherein the skin layer is composed of polypropylene. ### Claim 16 (depends on 14) The film of claim 14 wherein the skin layer is composed of polyethylene. ### Claim 17 (depends on 14) The film of claim 14 wherein the skin layer has a thickness within the range of 0.0001 inches to about 0.002 inches. ### Claim 18 (depends on 17) The film of claim 17 wherein the skin layer has a thickness of about 0.0005 inches. ### Claim 19 (depends on 1) The film of claim 1 wherein the first layer has an electrostatic charge greater than 40 dynes/cm. ### Claim 20 A multi-layer, flexible, gas-permeable film suitable for forming a cell culture container, the film comprising: a first layer composed of a polystyrene having a thickness within the range of 0.0001 inches to about 0.0010 inches, the first layer defining a cell growth surface; a second layer adhered to the first layer composed of a polymer alloy blend having multiple components; and, wherein the film having physical properties within the range: a>10,000 but <30,000 b>9 but <15 c>40 but <80 d>10 but <100 e<20 wherein: a is the flexural modulus in psi of the film measured according to ASTM D-790; b is the oxygen permeability in Barrers; c is the carbon dioxide permeability in Barrers; d is the nitrogen permeability in Barrers; and e is the water vapor transmission rate in (g mil/100 in 2/day). ### Claim 21 (depends on 20) The film of claim 20 wherein the film has an optical clarity within the range of 0.1%-10% as measured by a Hazometer under ASTM D1003. ### Claim 22 (depends on 20) The film of claim 20 wherein the polymer alloy has three components, a first component is styrene-ethylene-butadiene-styrene block copolymer, a second component is ethylene vinyl acetate, and a third component is polypropylene. ### Claim 23 (depends on 22) The film of claim 22 wherein the styrene-ethylene-butadiene-styrene block copolymer constitutes 40%-85% by weight of the polymer alloy, the ethylene vinyl acetate constitutes 0 to 40%% by weight of the polymer alloy, and the polypropylene constitutes 10 to 40% by weight of the polymer. ### Claim 24 (depends on 20) The film of claim 20 wherein the second layer is composed of a four component polymer alloy. ### Claim 25 (depends on 24) The film of claim 24 wherein the four component polymer alloy has a first component of a polypropylene, a second component selected from the group consisting essentially of an ultra low density polyethylene and polybutene-1, a third component of a dimer fatty acid polyamide, and a fourth component of a styrene-ethylene-butadiene-styrene block copolymer. ### Claim 26 (depends on 25) The film of claim 25 wherein the first component constitutes within the range of 30-60% by weight of the polymer alloy, the second component constitutes within the range of 25%-50% by weight of the polymer alloy, the third component constitutes within the range of 5%-40% by weight of the polymer alloy and the fourth component constitutes 5%-40% by weight of the polymer alloy. ### Claim 27 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26) A cell culture container comprising a first and second side wall each having an interior surface that cooperate to define a containment area, wherein at least the first side walls composed of a film of any of claims 1-26. ### Claim 28 (depends on 27) The container of claim 27 wherein the polystyrene first layer faces the containment area. ### Claim 29 A flexible, gas-permeable cell culture container suitable for culturing cells, the container comprising: a first and second side wall each having edges, the first and second side walls being sealed together at their respective side wall edges to provide a containment area, wherein at least the first side wall is composed of a first layer of polystyrene having a thickness within the range of 0.0001 inches to a out 0.0010 inches the first layer facing an interior of the container to define a cell growth surface, and, a second layer adhered to the first layer of a polyolefin, the second layer having thickness within the range of 0.004 inches to about 0.015 inches. ### Claim 30 (depends on 29) The cell culture container of claim 29 further comprising at least one access port for accessing the containment area. ### Claim 31 A method for fabricating a multi-layered film suitable for forming a container for culturing cells comprising the steps of: providing a polystyrene cell growth surface; providing a polymer alloy blend having multiple components; and, coextruding the polystyrene and the polymer alloy blend producing a layered film having a gas permeability to promote cell growth. ### Claim 32 (depends on 31) The method of claim 31 wherein the polystyrene layer has a thickness within the range of about 0.0001 to about 0.0010 inches, and wherein the polyolefin layer has a thickness within the range of about 0.004 to about 0.015 inches. ### Claim 33 A flexible, gas-permeable cell culture container suitable for culturing cells, the container comprising: a first side wall of the container being suitable for growing adherent cells, the first side wall comprises a first layer of polystyrene having a thickness within the range of 0.0001 inches to about 0.0010 inches, and, a second layer adhered to the first layer of a polymer alloy having multiple components, the second layer having a thickness within the range of 0.004 inches to about 0.015 inches; a second side wall attached to the first side wall for growing non-adherent cells; and, means associated with the container for distinguishing the first side wall from the second side wall. ### Claim 34 (depends on 33) The container of claim 33 wherein the means for distinguishing the first side wall from the second side wall is the geometry of the container. ### Claim 35 A method for culturing cells comprising the steps of: providing a flexible cell culture container having at least one side wall of a film having a first layer of a polystyrene having a thickness from 0.0001 inches to about 0.0010 inches, and a second layer adhered to the first layer of a polymeric material having a thickness from 0.004 inches to about 0.015 inches, the first layer faces an interior of the container to provide a cell growth surface; adding to the container a cell growth medium; and seeding the container with cells to be grown. ### Claim 36 (depends on 35) The method of claim 35 wherein the second layer is a polymer alloy having multiple components. ### Claim 37 (depends on 36) The film of claim 36 wherein the polymer alloy has three components, a first component is a styrene-ethylene-butadiene-styrene block copolymer, a second component is ethylene vinyl acetate, and a third component is polypropylene. ### Claim 38 (depends on 37) The film of claim 37 wherein the styrene-ethylene-butadiene-styrene block copolymer constitutes 40 to 85% by weight of the polymer alloy, the ethylene vinyl acetate constitutes 0 to 40% by weight of the polymer alloy, and the polypropylene constitutes 0 to 40% by weight of the polymer alloy. ### Claim 39 (depends on 36) The film of claim 36 wherein the second layer is composed of a four component polymer alloy. ### Claim 40 (depends on 39) The film of claim 39 wherein the four component polymer alloy has a first component of a polypropylene, a second component selected from the group consisting of an ultra low density polyethylene and polybutene-1, a third component of a dimmer fatty acid polyamide, and a fourth component of a styrene-ethylene-butadiene-styrene block copolymer. ### Claim 41 (depends on 40) The film of claim 40 wherein the first component constitutes within the range of 30-60% by weight of the polymer alloy, the second component constitutes within the range of 25%-50% by weight of the polymer alloy, the third component constitutes within the range of 5%-40% by weight of the polymer alloy, and the fourth component constitutes 5%-40% by weight of the polymer alloy. ### Claim 42 (depends on 35) The film of claim 35 further comprising a tie layer interposed between the first and second layers, the tie layer providing adhesive compatibility between the first and second layers.