# US 7,608,447 B2 · Pulse-medium perfusion bioreactor with improved mass transport for multiple-3-D cell constructs > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 7,608,447 B2 - Document ID: US7608447B2 - Status: Granted - Kind: B2 - Application number: 10/581,848 - Filed: 2004-12-09 - Published: 2007-05-17 - Granted: 2009-10-27 - Earliest claimed: 2004-12-09 - Assignees: Ben-Gurion University of the Negev Research and Development Authority - Inventors: Smadar Cohen; Tal Dvir; Michal Shachar - Web page: https://patentgrep.com/patent/US7608447B2 ## CPC - C12M 25/14: {Scaffolds; Matrices (in general C12N5/0068)} - C12M 29/10: {Perfusion} - C12M 41/18: {Heat exchange systems, e.g. heat jackets or outer envelopes} - 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 Disclosed is a novel net member for supporting one or more cell constructs in the culture chamber of a bioreactor, which comprises an array of impermeable pyramidal elements protruding from the face thereof, wherein each of the corners of the base of each of said impermeable pyramidal elements comprises a circular opening. This net member provides for uniform and unobstructed flow of culture medium through 3D cell constructs in culture. Also disclosed are a bioreactor and bioreactor system incorporating said net member. The bioreactor and bioreactor system may be used in the bioproduction of therapeutic proteins and stem cell expansion. ## Claims ### Claim 1 A net for supporting one or more cell constructs in the culture chamber of a bioreactor, comprising an array of impermeable pyramidal elements protruding from the face of said net, wherein each of the corners of the base of each of said impermeable pyramidal elements comprises a circular opening. ### Claim 2 (depends on 1) The net according to claim 1, wherein the diameter of said circular opening is between 0.1 mm to 3 mm. ### Claim 3 (depends on 1) The net according to claim 1, wherein the distance between any two adjacent circular openings situated orthogonally to one another along the x- or y-axis is between 1 mm to 10 mm. ### Claim 4 (depends on 1) The net according to claim 1, wherein the angle of the outer edges of the pyramidal elements is between 1° to 179°. ### Claim 5 (depends on 1) The net according to claim 1, wherein said net is constructed from a material selected from the group consisting of poly(methyl methacrylate), polycarbonate or any solid transparent material. ### Claim 6 A bioreactor comprising an inlet half having an opening at one end and an inlet aperture at its opposite end, and an outlet half having an opening at one end and an outlet aperture at its opposite end, wherein said halves are joined at their opening ends such that the hollow interior of said bioreactor forms a culture chamber, and further comprising at least one net for supporting one or more cell constructs in the culture chamber of a bioreactor, comprising an array of impermeable pyramidal elements protruding from the face of said net, wherein each of the corners of the base of each of said impermeable pyramidal elements comprises a circular opening, wherein said net is positioned transversely within said culture chamber. ### Claim 7 (depends on 6) The bioreactor according to claim 6, wherein said culture chamber is cylindrical in shape. ### Claim 8 (depends on 7) The bioreactor according to claim 7, wherein said net is circular. ### Claim 9 (depends on 7) The bioreactor according to claim 7, wherein the diameter of said net is equal to the diameter of said culture chamber. ### Claim 10 (depends on 6) The bioreactor according to claim 6, wherein two identical said nets are positioned transversely within said culture chamber. ### Claim 11 (depends on 10) The bioreactor according to claim 10, wherein at least one of said nets is permanently affixed to the circumference of said culture chamber. ### Claim 12 (depends on 10) The bioreactor according to claim 10, comprising means for removably affixing at least one of said nets within said culture chamber. ### Claim 13 (depends on 12) The bioreactor according to claim 12, wherein said means comprises a projection which protrudes inward from the circumference of said culture chamber wall. ### Claim 14 (depends on 6) The bioreactor according to claim 6, wherein said inlet aperture is threaded for suitably attaching means for transferring a fluid medium to said culture chamber. ### Claim 15 (depends on 6) The bioreactor according to claim 6, wherein said outlet aperture is suitably threaded for attaching means for transferring fluid medium from said culture chamber. ### Claim 16 (depends on 14) The bioreactor according to claim 14, wherein said means comprise transfer tubing. ### Claim 17 (depends on 6) The bioreactor according to claim 6, wherein said inlet half and said outlet half are joined to each other via screws or bolts. ### Claim 18 (depends on 6) The bioreactor according to claim 6, wherein the means for sealing said inlet half with said outlet half comprises an O-ring. ### Claim 19 (depends on 6) The bioreactor according to claim 6, wherein a fluid distributor mesh is positioned in said inlet half between said inlet aperture and said net. ### Claim 20 (depends on 19) The bioreactor according to claim 19, wherein a fluid distributor mesh is positioned in said outlet half between said outlet aperture and said net. ### Claim 21 (depends on 20) The bioreactor according to claim 20, wherein any of said fluid distributor meshes comprise pores whose diameter is up to 10 mm. ### Claim 22 (depends on 6) The bioreactor according to claim 6, wherein said bioreactor may be used for bioproduction of therapeutic protein. ### Claim 23 (depends on 6) The bioreactor according to claim 6, wherein said bioreactor may be used for stem cell expansion. ### Claim 24 A bioreactor system comprising: a. a bioreactor comprising an inlet halt having an opening at one end and an inlet aperture at its opposite end, and an outlet half having an opening at one end and an outlet aperture at its opposite end, wherein said halves are joined at their opening ends such that the hollow interior of said bioreactor forms a culture chamber, and comprising two identical nets for supporting at least one cell construct in said chamber, wherein the distance between said nets is equal to the thickness of said at least one cell construct, wherein each of said nets comprises an array of pyramidal elements protruding from the face of said net, wherein the vertex of each of said pyramidal elements comprises a circular opening b. a culture medium reservoir for storing a supply of a fluid culture medium, comprising a medium inlet and outlet, and further comprising a gas inlet and outlet; c. a gas supply for supplying gas to said medium contained in said reservoir, d. a heat exchanger for maintaining the temperature of said medium at a constant value, and e. a pump for pumping said medium from said reservoir into said bioreactor and back to said reservoir. ### Claim 25 (depends on 24) The bioreactor system according to claim 24, wherein said reservoir comprises a medium sample collection outlet. ### Claim 26 (depends on 24) The bioreactor system according to claim 24, wherein said pump is a peristaltic pump. ### Claim 27 A method for the cultivation of 3-D cell constructs, comprising the following steps: a. providing a bioreactor system comprising i. a bioreactor, said bioreactor comprising an inlet half having an opening at one end and an inlet aperture at its opposite end, and an outlet half having an opening at one end and an outlet aperture at its opposite end, wherein said halves are joined at their opening ends such that the hollow interior of said bioreactor forms a culture chamber, and comprising two identical nets for supporting at least one cell construct in said chamber, wherein the distance between said nets is equal to the thickness of said at least one cell construct, wherein each of said nets comprises an array of pyramidal elements protruding from the face of said net, wherein the vertex of each of said pyramidal elements comprises a circular opening; ii. a culture medium reservoir for storing a supply of a fluid culture medium, comprising a medium inlet and outlet; iii. a gas supply for supplying gas to said medium contained in said reservoir; iv. a heat exchanger for maintaining the temperature of said medium at a constant value; and v. a pump for pumping said medium from said reservoir into said bioreactor and back to said reservoir; b. placing at least one cell construct within said culture chamber, between said nets; c. pumping said medium from said reservoir into said culture chamber, thereby causing medium perfusion into said cell construct for a suitable period of time; and d. harvesting the resulting construct. ### Claim 28 (depends on 27) The method according to claim 27, further comprising: e. removing a sample of said medium from said reservoir after step c and before step d, in order to determine whether said medium should be replaced with new medium; f. adding fresh medium to said reservoir when necessary, after step e and before step d. ### Claim 29 (depends on 27) The method according to claim 27, wherein said cell construct consists of a polymeric scaffold seeded with cells. ### Claim 30 (depends on 29) The method according to claim 29 wherein said polymer is a polysaccharide. ### Claim 31 (depends on 29) The method according to claim 29, wherein said cells are human cells. ### Claim 32 (depends on 27) The method according to claim 27, wherein said 3-D cell constructs are for the bioproduction of therapeutic proteins. ### Claim 33 (depends on 27) The method according to claim 27, wherein said 3-D cell constructs are for stem cell expansion. ### Claim 34 (depends on 2) The net according to claim 2 wherein the diameter of said circular opening is 1.25 mm. ### Claim 35 (depends on 3) The net according to claim 3, wherein the distance between any two adjacent circular openings situated orthogonally to one another along the x- or y-axis is 3 mm. ### Claim 36 (depends on 4) The net according to claim 4, wherein the angle of the outer edges of the pyramidal elements is 60°. ### Claim 37 (depends on 21) The bioreactor according to claim 21, wherein any of said fluid distributor meshes comprise pores whose diameter is 2 mm. ### Claim 38 (depends on 30) The method according to claim 30 wherein the polysaccharide is alginate. ### Claim 39 (depends on 31) The method according to claim 31 wherein the human cells are cardiomyocytes.