# US 2011/0003390 A1 · Method and device for cell culture in open continuous mode > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 2011/0003390 A1 - Document ID: US20110003390A1 - Status: Published application - Kind: A1 - Application number: 12/918,987 - Filed: 2009-02-20 - Published: 2011-01-06 - Earliest claimed: 2008-02-21 - Assignees: Eco Solution - Inventors: Bernard KUDLA - Web page: https://patentgrep.com/patent/US20110003390A1 ## CPC - C12M 47/04: {Cell isolation or sorting (purging biological preparations of unwanted cells C12N5/0081, determining the presence or kind of microorganism C12Q1/04)} - 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 The present invention relates to the use of cell cultures in the open continuous mode, to a method for selecting static cell variants or cell variants which proliferate in suspension, to a culture substrate and to a device suitable for implementing this method. ## Claims ### Claim 1 Method for continuous cell culture allowing the selection of cell variants that are static or proliferating in suspension, characterized in that it comprises the following stages: a) a liquid culture medium contained in a first culture vessel kept open in a closed container is seeded using one or more live cells, b) said cells, in said culture medium, are brought to a specific growth stage, corresponding to a given cell density or to a physicochemical parameter measured in the culture medium, c) the cell density in the culture medium, or the value of said physicochemical parameter, reached in stage b), is kept substantially constant by supplying fresh culture medium or a diluent in said culture vessel, d) a portion of the culture medium of the culture obtained in c) containing the cells in suspension is removed by pipetting so as to maintain the volume of the culture, e) a fraction of the culture medium obtained in d) is transferred into a second culture vessel, f) said first culture vessel with the remaining culture fraction that it contains is withdrawn; g) after several generations of culture in the second vessel, the cells proliferating in suspension and/or the static cell variants are selected. ### Claim 2 Method for continuous cell culture allowing the selection of cell variants proliferating in suspension, characterized in that it comprises the following stages: a) a liquid culture medium contained in a first culture vessel kept open in a closed container is seeded using one or more live cells, b) the cells, in said culture medium, are brought to a specific growth stage, corresponding to a given cell density or to a physicochemical parameter measured in the culture medium, c) the cell density of the culture, or the value of said physicochemical parameter, reached in stage b), is kept substantially constant by supplying fresh culture medium or at least one diluent in said culture vessel, d) a portion of the culture medium containing the cells in suspension is removed by pipetting so as to maintain the volume of the culture, e) a fraction of the culture obtained in d) in which the cells are in suspension, is transferred to a second culture vessel replacing the first; f) said first culture vessel with the remaining culture fraction that it contains is withdrawn; g) after several generations of culture in the second vessel, the cells proliferating in suspension are selected. ### Claim 3 Method for continuous cell culture allowing the selection of static cell variants, characterized in that it comprises the following stages: a) a liquid culture medium contained in a first culture vessel is seeded using one or more live cells, in which at least one solid surface, preferably a plate, is placed, said vessel being kept open in a closed container, b) the cells, in the seeded culture medium, are brought to a specific growth stage, corresponding to a given cell density or to a physicochemical parameter measured in the culture medium, c) the cell density of the culture, or the value of said physicochemical parameter, reached in stage b), is kept substantially constant by supplying fresh culture medium or at least one diluent in said culture vessel, d) a portion of the culture medium obtained in c) containing the cells in suspension is removed by pipetting so as to maintain the volume of the culture, e) said solid surface on which a fraction of the culture obtained in d) is deposited, is transferred to a second culture vessel replacing the first, f) said first culture vessel with the remaining fraction of culture that it contains is withdrawn, g) after several generations of culture, the static cell variants adhering to said solid surface are selected. ### Claim 4 (depends on 3) Method according to claim 3, characterized in that said solid surface is made up of a material treated in order to avoid adherence of the cells. ### Claim 5 (depends on 1, 2, 3, 4) Method according to any one of claims 1 to 4, characterized in that said culture vessels are kept open at the top, and that a gas stream, such as sterile air, is applied continuously at the periphery of their opening. ### Claim 6 (depends on 1, 2, 3, 4, 5) Method according to any one of claims 1 to 5, characterized in that a sterile gas stream is applied from the top to the bottom of the culture vessels. ### Claim 7 Method according to the previous claim, characterized in that the sterile gas stream from the top to the bottom is maintained by creating a partial vacuum at the periphery of the culture vessel. ### Claim 8 (depends on 7) Method according to claim 7, characterized in that the sterile gas stream is discharged out of the container at the bottom of said culture vessels. ### Claim 9 (depends on 1, 2, 3, 4, 5, 6, 7, 8) Method according to any one of claims 1 to 8, characterized in that at least the different stages a) to f) are carried out in a closed container. ### Claim 10 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9) Method according to any one of claims 1 to 9, characterized in that stages a) to f) are reiterated one or more times before proceeding with stage g). ### Claim 11 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) Method according to any one of claims 1 to 10, characterized in that a substantially constant cell density is maintained in the culture in stage c) by diluting the culture with fresh medium while keeping a constant volume of culture medium in the culture vessel. ### Claim 12 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11) Method according to any one of claims 1 to 11, characterized in that the transfer of the culture medium containing the cells in suspension to the second culture vessel is carried out by pipetting using a sterile pipette. ### Claim 13 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) Method according to any one of claims 1 to 12, characterized in that the first used culture vessel is discharged from the closed container using an airlock. ### Claim 14 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13) Method according to any one of claims 1 to 13, characterized in that the culture vessel in operation is placed in a thermostatically controlled cell allowing regulation of the temperature of the cell culture. ### Claim 15 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14) Method according to any one of claims 1 to 14, characterized in that several cultures are carried out simultaneously in several culture supports placed in the same container. ### Claim 16 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15) Method according to any one of claims 1 to 15, characterized in that sampling of the culture is carried out regularly. ### Claim 17 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16) Method according to any one of claims 1 to 16, characterized in that the culture supports or the culture vessels are single-use. ### Claim 18 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17) Method according to any one of claims 1 to 17, characterized in that gas is injected into the cells in culture by means of a bubbling device introduced into the culture vessel. ### Claim 19 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18) Method according to any one of claims 1 to 18, characterized in that several stages of the method are carried out using one or more automated arms allowing movements inside the container. ### Claim 20 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19) Method according to any one of claims 1 to 19, characterized in that the cells are cultured continuously over a number of generations greater than 102, preferably greater than 104, more preferentially greater than 106, and even more preferentially greater than 1010 generations, without opening of the container to the outside environment. ### Claim 21 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20) Method according to any one of claims 1 to 20, characterized in that it is applied to the culture of autotrophic microorganisms and that said culture is illuminated during the different stages of said method. ### Claim 22 Culture support (18) making it possible to carry out a cell culture in open continuous mode, characterized in that it comprises: at least one culture vessel (1) open at the top (6) suitable for containing a liquid culture medium; at least one containment tank (2) open towards the top, in which said culture vessel is housed (1); a space (7) between said culture vessel (1) and said containment tank (2), suitable for allowing a gas stream to circulate at the periphery of the opening of the vessel, from the top to the bottom, between the culture vessel (1) and the inner walls of the containment tank (2); and a means for extracting (8) said gas stream, situated in the lower part of the containment tank (2). ### Claim 23 (depends on 22) Culture support according to claim 22, characterized in that said means for extracting the gas stream consists of one or more openings (8) allowing passage of the gas stream. ### Claim 24 (depends on 22, 23) Culture support according to claim 22 or 23, characterized in that it is presented in the form of a removable block (12) in which several of said containment tanks are grouped together and in which at least one of said culture vessels (1) is housed. ### Claim 25 (depends on 22, 23, 24) Culture support according to one of claims 22 to 24, characterized in that said lower part of the containment tank (2) is embedded in a base provided with additional means (14) for extracting said gas stream circulating between the culture vessels (1) and the walls of the containment tanks (2), such as a pipe connected to a vacuum pump, or one or more gas distribution means. ### Claim 26 (depends on 22, 23, 24, 25) Culture support according to one of claims 22 to 25, characterized in that it also comprises at least one means for regulating the temperature (3) of the internal volume of said containment tank (2). ### Claim 27 (depends on 26) Support according to claim 26, characterized in that said means for regulating the temperature of the internal volume (7) of said containment tank consists of a heating resistance enclosed in at least one of the walls of said containment tank (2). ### Claim 28 (depends on 22, 23, 24, 25, 26, 27) Culture support according to any one of claims 22 to 27, characterized in that it also comprises at least one means for injecting air into the culture medium (media), contained in said vessel(s), such as one or more aeration rods (4). ### Claim 29 (depends on 22, 23, 24, 25, 26, 27, 28) Culture support according to any one of claims 22 to 28, characterized in that it also comprises at least one means for optically measuring (9) of the cell density present in the culture medium contained by the culture vessel (1). ### Claim 30 (depends on 22, 23, 24, 25, 26, 27, 28, 29) Culture support according to any one of claims 22 to 29, characterized in that it also comprises at least one means for producing light (15) for the culture of microorganisms in autotrophic mode, located in the space between the culture vessel and the inner walls of said containment tank (2), or enclosed in one of the walls of said containment tank. ### Claim 31 (depends on 22, 23, 24, 25, 26, 27, 28, 29, 30) Cell culture device allowing a continuous growth of the cells in open mode, characterized in that it comprises: a container (17); a means for generating a sterile gas stream (23) passing through said container; one or more culture supports (18) according to any one of claims 22 to 30, positioned in said container (17), making it possible to carry out a cell culture in open mode. ### Claim 32 (depends on 31) Cell culture device according to claim 31, characterized in that it also comprises: at least one means for replenishing the culture medium placed inside said container. ### Claim 33 (depends on 31, 32) Cell culture device according to claim 31 or 32, characterized in that it also comprises: at least one culture vessel (1) suitable for replacing that contained in the culture support (18). ### Claim 34 (depends on 31, 32, 33) Device according to any one of claims 31 to 33, characterized in that the means for generating the sterile gas stream (23) is placed in the upper part of the container (17) so that said sterile gas stream is directed from the top to the bottom of the culture support (18). ### Claim 35 (depends on 31, 32, 33, 34) Cell culture device according to any one of claims 31 to 34, characterized in that it also comprises at least one means for extracting the sterile gas stream (8) at the bottom of said culture vessel. ### Claim 36 (depends on 35) Device according to claim 35, characterized in that the means for extracting the sterile gas stream (8) creates a partial vacuum in the space (7) situated between the culture vessel (1) and the containment tank (2) of the culture support (18). ### Claim 37 (depends on 35, 36) Device according to claim 35 or 36, characterized in that said means for extracting (8) is associated with at least one means for aspirating the air suitable for sucking out the air surrounding the periphery of the opening (6) of the culture vessel. ### Claim 38 (depends on 32, 33, 34, 35, 36, 37) Device according to any one of claims 32 to 37, characterized in that the means for replenishing the culture medium comprises a means for transferring (20) part of the contents of the culture vessel to a discharge zone. ### Claim 39 (depends on 32, 33, 34, 35, 36, 37, 38) Device according to any one of claims 32 to 38, characterized in that the means for replenishing the culture medium comprises a means for transferring (20) fresh medium from a reserve situated inside the container (17) to the culture vessel. ### Claim 40 (depends on 38, 39) Device according to one of claim 38 or 39, characterized in that the means for transferring comprises a pipette (20) and a means for aspirating the fresh or used medium in said pipette. ### Claim 41 (depends on 31, 32, 33, 34, 35, 36, 37, 38, 39, 40) Device according to one of claims 31 to 40, characterized in that it also comprises: at least one outlet means to the outside of the container of the means for transferring (20) and/or of at least one culture vessel. ### Claim 42 (depends on 41) Device according to claim 41, characterized in that said outlet means comprises an airlock. ### Claim 43 (depends on 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42) Device according to any one of claims 31 to 42, characterized in that it comprises at least one means for injecting air (4, 21) into the culture. ### Claim 44 (depends on 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43) Device according to any one of claims 31 to 43, characterized in that it comprises at least one automated arm (19) suitable for carrying out movements inside the container. ### Claim 45 (depends on 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44) Device according to any one of claims 31 to 44, characterized in that the container (17) is closed. ### Claim 46 (depends on 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45) Device according to any one of claims 31 to 45, characterized in that it comprises several culture supports positioned in said container, for carrying out several cell cultures in open mode in parallel. ### Claim 47 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46) Device according to any one of claims 30 to 46, characterized in that it allows the implementation of the method according to any one of claims 1 to 21.