# US 11,999,969 B2 · Methods of cell culture for adoptive cell therapy > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 11,999,969 B2 - Document ID: US11999969B2 - Status: Granted - Kind: B2 - Application number: 18/511,208 - Filed: 2023-11-16 - Published: 2024-03-14 - Granted: 2024-06-04 - Earliest claimed: 2009-12-08 - Assignees: Wilson Wolf Manufacturing; Baylor College of Medicine - Applicants: Wilson Wolf Manufacturing; Baylor College of Medicine - Inventors: Juan F. Vera; Cliona M. Rooney; Ann M. Leen; John R. Wilson - Web page: https://patentgrep.com/patent/US11999969B2 ## CPC - C12M 23/24: {Gas permeable parts} - A61K 39/0011: {Cancer antigens} - C12N 5/0638: {Cytotoxic T lymphocytes [CTL] or lymphokine activated killer cells [LAK]} - A61K 2039/5156: {expressing foreign proteins} - A61K 2039/5158: {Antigen-pulsed cells, e.g. T-cells} - C07K 14/7051: {T-cell receptor (TcR)-CD3 complex} - C07K 2319/03: containing a transmembrane segment - C12N 2501/2302: Interleukin-2 (IL-2) - C12N 2502/11: blood or immune system cells - 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} - A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D) - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00) ## Abstract An improved method of culturing cells for cell therapy applications that includes growing desired cells in the presence of antigen-presenting cells and/or feeder cells and with medium volume to surface area ratio of up to 1 ml/cm2 if the growth surface is not comprised of gas permeable material and up to 2 ml/cm2 if the growth surface is comprised of gas permeable material. The desired cells are at a surface density of less than 0.5×106 cells/cm2 at the onset of a production cycle, and the surface density of the desired cells plus the surface density of the antigen presenting cells and/or feeder cells are at least about 1.25×105 cells/cm2. ## Claims ### Claim 1 A method for culturing and then concentrating cells in a gas permeable cell culture and cell recovery device comprising: adding an initial volume of medium and cells into the gas permeable cell culture and cell recovery device; said gas permeable cell culture and cell recovery device comprising a cell culture compartment bounded by a growth surface and a wall that is perpendicular to said growth surface, said growth surface comprised of liquid impermeable gas permeable material, the bottom of said gas permeable material in contact with a growth surface support that holds the gas permeable material in a horizontal position during culture and allows ambient gas to contact the underside of the gas permeable material by random motion, said wall is more than 2.0 cm in height; wherein the initial volume of media resides at an initial medium height determined by the distance from the lowermost medium location to the uppermost medium location; and wherein the initial medium height is beyond 2.0 cm, and the ratio of said initial volume of media to the growth surface area is beyond 2.0 mL/cm2 and the medium is not perfused; allowing the cells to gravitate to the growth surface and allowing the cells to reside in a state of static culture without medium replenishment for a period of time with said gas permeable cell culture and cell recovery device in a location with ambient gas suitable for cell culture such that the cells expand; concentrating the cells by inserting a medium removal conduit into the gas permeable cell culture and cell recovery device and removing medium to obtain a cell recovery medium volume and a cell recovery medium height, wherein the cell recovery medium height is 0.2 cm or more, and the cell recovery volume to growth surface area ratio is 0.2 ml/cm2 or more. ### Claim 2 (depends on 1) The method of claim 1, further comprising a step of mixing the cells in the cell recovery medium volume, such that cells are distributed within the cell recovery medium volume and withdrawing the cell recovery medium with the medium removal conduit. ### Claim 3 (depends on 1) The method of claim 1, further comprising a step of mixing the cells in the cell recovery medium volume, such that cells are distributed within the cell recovery medium volume, reorienting the gas permeable cell culture and cell recovery device into a position at an angle that deviates from the original horizontal position in order to relocate the cell recovery medium volume, and withdrawing the cell recovery medium with the medium removal conduit. ### Claim 4 (depends on 1) The method of claim 1 wherein the ratio of the initial volume of medium to the growth surface area is at least 2.5 mL/cm2. ### Claim 5 (depends on 1) The method of claim 1 wherein the device is clear to allow visual assessment. ### Claim 6 (depends on 1) The method of claim 1 wherein the uppermost medium location is parallel to the growth surface. ### Claim 7 (depends on 1) The method of claim 1 wherein said gas permeable material is non-porous. ### Claim 8 (depends on 1) The method of claim 1 wherein an initial volume of medium, cells, and cytokine are added into the gas permeable cell culture and cell recovery device. ### Claim 9 (depends on 8) The method of claim 8 wherein the cytokine is IL2. ### Claim 10 (depends on 1) The method of claim 1 wherein the cells are T cells. ### Claim 11 (depends on 1) The method of claim 1, wherein said gas permeable material is comprised of silicone. ### Claim 12 (depends on 1) The method of claim 1 wherein the cells are CAR-T cells. ### Claim 13 (depends on 1) The method of claim 1 wherein the cells are PBMCs. ### Claim 14 (depends on 1) The method of claim 1 wherein the cells are effector cells and feeder cells. ### Claim 15 (depends on 14) The method of claim 14 wherein the feeder cells are gamma-irradiated antigen presenting cells, K562, or LCLs. ### Claim 16 (depends on 14) The method of claim 14 wherein the effector cells are T cells, NK, Treg, TIL, or MIL. ### Claim 17 (depends on 14) The method of claim 14 wherein the effector cells are antigen specific T cells. ### Claim 18 (depends on 1) The method of claim 1 wherein the wall is rigid. ### Claim 19 (depends on 1) The method of claim 1 wherein the gas permeable cell culture and cell recovery device includes a cap or a cover. ### Claim 20 (depends on 1) The method of claim 1 wherein the medium removal conduit is a pipette.