# US 2014/0196791 A1 · Method and apparatus for the use of micro-carriers in a disposable bioreactor system > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 2014/0196791 A1 - Document ID: US20140196791A1 - Status: Published application - Kind: A1 - Application number: 14/153,679 - Filed: 2014-01-13 - Published: 2014-07-17 - Earliest claimed: 2013-01-11 - Assignees: PBS Biotech, Inc. - Applicants: PBS BIOTECH, Inc. - Inventors: Chanyong Brian LEE; Yasunori HASHIMURA - Web page: https://patentgrep.com/patent/US20140196791A1 ## CPC - C12M 25/16: {Particles; Beads; Granular material; Encapsulation (chemical or physical processes conducted in the presence of fluids and solid particles B01J8/00)} - C12M 27/04: {with introduction of gas through the stirrer or mixing element} - C12M 27/06: {with horizontal or inclined stirrer shaft or axis} - C12M 29/04: {Filters; Permeable or porous membranes or plates, e.g. dialysis} - C12M 29/06: {Nozzles; Sprayers; Spargers; Diffusers (per se B01F23/231, B01J19/26)} - C12M 33/14: {with filters, sieves or membranes} - C12M 37/00: {Means for sterilizing, maintaining sterile conditions or avoiding chemical or biological contamination (C12M23/38 takes precedence; filtration in general and filters per se B01D24/00-B01D41/00; autoclaves B01J3/04; treatment of microorganisms with electrical or wave energy C12N13/00)} - C12M 41/00: {Means for regulation, monitoring, measurement or control, e.g. flow regulation (controlling or regulating chemical, physical or physicochemical processes B01J19/0006; heating or cooling apparatus for laboratory use B01L7/00; electro optical investigation of individual particles, flow cytometers G01N15/14; automatic analysis G01N35/00; controlling or regulating in general G06N)} - C12N 1/00: Microorganisms; Compositions thereof (medicinal preparations containing material from protozoa, bacteria or viruses A61K35/66, from algae A61K36/02, from fungi A61K36/06; preparing medicinal bacterial antigen or antibody compositions, e.g. bacterial vaccines, A61K39/00); Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor - Y10T 137/0318: Processes - 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} - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - Y10T: TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION ## Abstract A system and method for compartmentalizing micro-carriers in a bioreactor includes a container configured to store micro-carriers and a vessel configured to contain a fluid so as to prevent the micro-carriers from entering the vessel during sterilization, shipping, storage, or other pre-use handling of the system. One or more addition lines connect the container and the vessel such that the vessel is in fluid communication with the vessel. The one or more addition lines are contactable by at least one blocking element configured to reversibly block fluid flow between the container and the vessel. The container, addition lines, and vessel are configured to allow the micro-carriers to be injected into the vessel at any point during a cell culture run. The vessel may also include a rotatable wheel, a harvest port configured to allow for the removal of the micro-carriers from the vessel, and a media removal port comprising a retention screen for removing spent medium. ## Claims ### Claim 1 A bioreactor system, comprising: a micro-carrier container configured to store micro-carriers; a vessel configured to contain a fluid; and one or more fluid conduit lines connecting the micro-carrier container and the vessel such that the micro-carrier container is in fluid communication with the vessel, wherein the one or more fluid conduit lines is reversibly blocked to block fluid flow in the one or more lines, and wherein the micro-carrier container and the vessel are configured to allow the micro-carriers to be dropped into the vessel at any point during a cell culture procedure when the one or more fluid conduit lines are unblocked. ### Claim 2 (depends on 1) The bioreactor system according to claim 1, wherein the micro-carrier container comprises a pouch connected to the one or more fluid conduit lines. ### Claim 3 (depends on 1) The bioreactor system according to claim 1, wherein the one or more fluid conduit lines comprises a main addition line and the micro-carrier container terminates the main addition line, and wherein the system further comprises: a reversible flow blocking element disposed on the main addition line between the micro-carrier container and the vessel and configured to temporarily prevent micro-carriers from passing through the main addition line from the micro-carrier container to the vessel, and wherein the micro-carrier container, the vessel, the main addition line, and the blocking element are configured such that gravitational force is sufficient to cause micro-carriers to pass from the micro-carrier container to the vessel via the main addition line. ### Claim 4 (depends on 1) The bioreactor system according to claim 1, wherein the one or more fluid conduit lines comprises an addition line and the micro-carrier container is disposed in-line with the addition line, and wherein the system further comprises: a first reversible flow blocking element disposed on the addition line prior to the micro-carrier container and configured to control fluid flow into the micro-carrier container; and a second reversible flow blocking element disposed on the addition line between the micro-carrier container and the vessel and configured to control a flow of a fluid mixture comprising micro-carriers from the micro-carrier container to the vessel, wherein the micro-carrier container, the addition line, and the first reversible flow blocking element, are configured for selective addition of fluid into the micro-carrier container through a portion of the addition line so that micro-carriers contained in the micro-carrier container become dispersed in the fluid, and wherein the micro-carrier container, the addition line, the second reversible flow blocking element, and the vessel are configured for selective addition of a fluid mixture comprising micro-carriers from the micro-carrier container through a portion of the addition line and into the vessel. ### Claim 5 (depends on 1) The bioreactor system according to claim 1, wherein the one or more fluid conduit lines comprises a branched addition line and the micro-carrier container is disposed in-line with the branched addition line, and a primary addition line connected to the vessel, and wherein the system further comprises: a first reversible flow blocking element disposed on the branched addition line prior to the micro-carrier container and configured to control fluid flow into the micro-carrier container; a second reversible flow blocking element disposed on the branched addition line between the micro-carrier container and the vessel and configured to control a flow of a fluid mixture comprising micro-carriers from the micro-carrier container to the vessel; a third reversible flow blocking element disposed on the primary addition line and configured to control fluid flow through the primary addition line and into the vessel, wherein the branched addition line, the primary addition line, the first reversible flow blocking element, the third reversible flow blocking element, and the vessel are configured for selective addition of fluid into the vessel through the primary addition line while blocking fluid flow into the micro-carrier container, wherein the branched addition line, the primary addition line, the first reversible flow blocking element, the third reversible flow blocking element, and the micro-carrier container are configured for selective addition of fluid into the micro-carrier container through a portion of the branched addition line while blocking fluid flow through the primary addition line so that micro-carriers contained in the micro-carrier container become dispersed in the fluid, and wherein the branched addition line, the second reversible flow blocking element, the micro-carrier container, and the vessel are configured for selective addition of a fluid mixture comprising micro-carriers from the micro-carrier container through a portion of the branched addition line and into the vessel. ### Claim 6 (depends on 1) The bioreactor system according to claim 1, wherein the one or more fluid conduit lines is/are reversibly blocked by one or more clamps. ### Claim 7 (depends on 1) The bioreactor system according to claim 1, further comprising micro-carriers disposed within the micro-carrier container. ### Claim 8 (depends on 1) The bioreactor system according to claim 1, wherein the micro-carrier container, the vessel, the micro-carriers, and the one more fluid conduit lines are sterilized. ### Claim 9 (depends on 1) The bioreactor system according to claim 1, wherein the vessel comprises a curved bottom, and the system further comprises: a mixing wheel rotatably disposed within the vessel; at least one sparger port configured to inject gas into the vessel; a harvest port configured to allow for the removal of cultured cell material from the vessel; and a media removal port configured for removing fluid media from the vessel and comprising a retention screen, wherein the retention screen is configured to retain the micro-carriers in the vessel while fluid media is removed from the vessel via the media removal port. ### Claim 10 (depends on 9) The bioreactor of claim 9, wherein the mixing wheel is configured to be pneumatically driven and the at least one sparger port comprises a main sparger port configured to inject gas into the vessel below the mixing wheel to pneumatically drive rotation of the mixing wheel. ### Claim 11 (depends on 9) The bioreactor system according to claim 9, wherein the retention screen comprises a plurality of pores formed in a convex manner with respect to a fluid media side of the retention screen and having a perimeter that is raised toward an interior of the vessel. ### Claim 12 (depends on 1) The bioreactor system according to claim 1, wherein the vessel is a rigid or semi-rigid tank, or a flexible or partially flexible bag. ### Claim 13 (depends on 1) The bioreactor system according to claim 1, wherein the one or more fluid conduit lines comprise at least one aseptic connection device. ### Claim 14 (depends on 1) The bioreactor system according to claim 1, wherein the micro-carrier container comprises a segregatable portion of the one or more conduit lines. ### Claim 15 A method for compartmentalizing micro-carriers in a bioreactor system that comprises a micro-carrier container, a vessel, and one or more fluid conduit lines connecting the micro-carrier container and the vessel such that the micro-carrier container is in fluid communication with the vessel, the method comprising: placing the micro-carriers in the micro-carrier container so that the micro-carriers are separated from the vessel; reversibly blocking the fluid communication between the micro-carrier container and the vessel to retain the micro-carriers in the micro-carrier container; and sterilizing the vessel and the micro-carriers stored within the micro-carrier container. ### Claim 16 (depends on 15) The method according to claim 15, wherein the step of sterilizing comprises exposing the vessel and micro-carriers to sterilizing gamma radiation. ### Claim 17 (depends on 15) The method according to claim 15, further comprising aseptically packaging the sterilized vessel and the micro-carriers prior to adding fluid to the vessel. ### Claim 18 (depends on 15) The method according to claim 15, further comprising: adding a fluid to the vessel; reversing the fluid communication block between the micro-carrier container and the vessel; and moving the micro-carriers from the micro-carrier container into the vessel through the fluid communication between the micro-carrier container and the vessel. ### Claim 19 (depends on 15) The method according to claim 15, wherein the one or more fluid conduit lines comprise a main addition line connected to the vessel, the micro-carrier container terminates the main addition line, and the main addition line is reversibly blocked between the micro-carrier container and the vessel to temporarily prevent micro-carriers from passing through the main addition line from the micro-carrier container to the vessel, and wherein the method further comprises: adding a fluid to the vessel; reversing the reversible block in the main addition line between the micro-carrier container; and moving the micro-carriers from the micro-carrier container into the vessel via the main addition line between the micro-carrier container and the vessel. ### Claim 20 (depends on 19) The method of claim 19, wherein the micro-carriers are moved from the micro-carrier container to the vessel via gravity. ### Claim 21 (depends on 15) The method according to claim 15, wherein the one or more fluid conduit lines comprise an addition line, the micro-carrier container is disposed in-line with the addition line, the addition line is reversibly blocked prior to the micro-carrier container to control fluid flow into the micro-carrier container, and the addition line between the micro-carrier container and the vessel is reversibly blocked to control a flow of a fluid mixture comprising micro-carriers from the micro-carrier container to the vessel, and wherein the method further comprises: adding fluid to the vessel; reversing the reversible block in the addition line prior to the micro-carrier container; adding fluid to the micro-carrier container via the addition line, while maintaining the reversible block in the addition line between the micro-carrier container and the vessel; reversing the reversible block in the addition line between the micro-carrier container and the vessel; and moving a mixture comprising micro-carriers from the micro-carrier container into the vessel via the addition line between the micro-carrier container and the vessel. ### Claim 22 (depends on 15) The method according to claim 15, wherein the one or more fluid conduit lines comprises a branched addition line and a primary addition line connected to the vessel, the micro-carrier container is disposed in-line with the branched addition line, the branched addition line is reversibly blocked prior to the micro-carrier container to control fluid flow into the micro-carrier container, the branched addition line is reversibly blocked between the micro-carrier container and the vessel to control a flow of a fluid mixture comprising micro-carriers from the micro-carrier container to the vessel, and the primary addition line is reversibly blocked to control fluid flow through the primary addition line and into the vessel, and wherein the method further comprises: removing the reversible block in the primary addition line; adding fluid to the vessel via the primary addition line, while maintaining the reversible blocks in the branched addition line; removing the reversible block in the branched addition line prior to the micro-carrier container; adding fluid to the micro-carrier container via the branched addition line, while maintaining the reversible block in the branched addition line between the micro-carrier container and the vessel; removing the reversible block in the branched addition line between the micro-carrier container and the vessel; and moving a mixture comprising micro-carriers from the micro-carrier container into the vessel via the branched addition line between the micro-carrier container and the vessel. ### Claim 23 (depends on 15) The method according to claim 15, wherein the vessel comprises a curved bottom and the bioreactor system further comprises a mixing wheel rotatably disposed within the vessel, at least one sparger port configured to inject gas into the vessel, a harvest port configured to allow for the removal of cultured cell material from the vessel, and a media removal port configured for removing fluid media from the vessel and comprising a retention screen to retain the micro-carriers in the vessel while removing fluid media via the media removal port, and wherein the method further comprises: injecting a gas into the vessel via the at least one sparger port to pneumatically drive the rotatable mixing wheel, thereby creating a tangential flow of fluid between a periphery of the mixing wheel and the curved surface; and removing fluid from the vessel via the media removal port, while the retention screen retains micro-carriers in the vessel. ### Claim 24 (depends on 23) The method according to claim 23, further comprising removing the micro-carriers via the harvest port. ### Claim 25 (depends on 23) The method according to claim 23, wherein the bioreactor system further comprises one or more additional ports, and wherein the method further comprises injecting a fluid through the one or more additional ports to create a steady-state equilibrium of fluid volume within the vessel. ### Claim 26 (depends on 25) The method according to claim 25, wherein the fluid added to the micro-carrier container is a cell culture medium, a nutrient solution, phosphate buffered saline, or water.