# US 10,369,254 B2 · Method and apparatus for in vitro kidney organogenesis > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 10,369,254 B2 - Document ID: US10369254B2 - Status: Granted - Kind: B2 - Application number: 15/121,551 - Filed: 2015-02-06 - Published: 2016-12-15 - Granted: 2019-08-06 - Earliest claimed: 2014-02-26 - Assignees: The Regents of the University of California; U.S. Department of Veterans Affairs - Applicants: The Regents of the University of California; U.S. Department of Veterans Affairs - Inventors: Norimoto Yanagawa; Masaki Nishikawa; Morgan Hamon; Peter Viktor Hauser - Web page: https://patentgrep.com/patent/US10369254B2 ## CPC - A61L 27/3604: {characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel} - A61L 27/3683: {subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment} - C12M 21/08: {for producing artificial tissue or for ex-vivo cultivation of tissue (prostheses A61F2/00, grafts A61L27/00)} - C12M 23/16: {Microfluidic devices; Capillary tubes (integrated microfluidic structures B01L3/5027; microreactors B01J19/0093)} - C12M 23/58: {Reaction vessels connected in series or in parallel (combinations of bioreactors with other apparatus, C12M43/00)} - C12M 25/14: {Scaffolds; Matrices (in general C12N5/0068)} - C12N 5/0697: {Artificial constructs associating cells of different lineages, e.g. tissue equivalents (blood vessels C12N5/0691)} - C12N 2502/256: Renal cells - A61L: METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES (preservation of bodies or disinfecting characterised by the agents employed A01N; preserving, e.g. sterilising, food or foodstuffs A23; preparations for medical, dental or toiletry purposes A61K) - 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) ## Abstract The invention provides for a device, system, and methods for using the same with kidney progenitor cells, specifically ureteric bud (UB) cells, metanephric mesenchymal (MM) cells, and the stromal cell (SC) subpopulation of the metanephric mesenchyme, to generate an embryonic kidney organoid that can be implanted into a mammalian subject to create a living, functional kidney. ## Claims ### Claim 1 A method of generating an embryonic kidney organoid, the method comprising the steps of: providing a kidney mold device having a ureteric bud (UB) cell chamber and a metanephric mesenchymal (MM) cell chamber, wherein the MM cell chamber is fluidly connected to the UB cell chamber; providing a precursor micromold having a plurality of channels for generating a UB tube from UB cells, wherein the channels are dimensioned such that UB tubes formed in the channels fit within the UB cell chamber of the kidney mold device; seeding UB cells into the precursor micromold to generate a UB tube; transferring the UB tube into the kidney mold device UB cell chamber; generating a metanephric mesenchymal/stromal cell (MM/SC) aggregate or suspension; transferring the MM/SC aggregate or suspension into the kidney mold device MM cell chamber; and culturing the kidney mold device in growth media to generate an embryonic kidney organoid. ### Claim 2 (depends on 1) The method of claim 1, wherein said UB cell chamber is linearly shaped. ### Claim 3 (depends on 1) The method of claim 1, wherein said MM cell chamber has the shape of an ellipse. ### Claim 4 (depends on 1) The method of claim 1, wherein said MM cell chamber has the shape of a bulb. ### Claim 5 (depends on 1) The method of claim 1, wherein said MM cell chamber has the shape of a circle. ### Claim 6 (depends on 1) The method of claim 1, wherein said kidney mold device is formed from a hydrogel. ### Claim 7 (depends on 1) The method of claim 1, wherein said kidney mold device is formed from a plastic polymer. ### Claim 8 (depends on 1) The method of claim 1, wherein said UB cell chamber and said MM cell chamber are incorporated into a microfluidic device. ### Claim 9 (depends on 1) The method of claim 1, wherein the kidney progenitor UB cells and MM/SC aggregate or suspension comprise embryonic cells differentiated towards the renal lineages of UB, MM, and stromal cells (SC). ### Claim 10 (depends on 1) The method of claim 1, wherein the kidney progenitor UB cells and MM/SC aggregate or suspension comprise induced pluripotent stem cells differentiated towards the renal lineages of UB, MM, and SC. ### Claim 11 (depends on 1) The method of claim 1, wherein the UB cells and MM/SC aggregate or suspension comprise urine derived stem cells differentiated towards the renal lineages of UB, MM, and SC. ### Claim 12 (depends on 1) The method of claim 1, wherein the cells and MM/SC aggregate or suspension comprise amniotic fluid stem cells differentiated towards the renal lineages of UB, MM, and SC. ### Claim 13 (depends on 1) The method of claim 1, further comprising the step of implanting said embryonic kidney organoid into a mammalian subject so that it can mature into a functional kidney. ### Claim 14 (depends on 13) The method of claim 13, wherein said embryonic kidney organoid is a human embryonic kidney organoid. ### Claim 15 (depends on 1) The method of claim 1, wherein the MM/SC are suspended in a medium selected from the group consisting of collagen, agarose, polyacrylamide, and matrigel. ### Claim 16 (depends on 1) The method of claim 1, wherein the MM/SC are aggregated into a sphere by overnight culture or by centrifugation. ### Claim 17 (depends on 1) The method of claim 1, wherein extracellular matrix (ECM) of an embryonic or adult kidney is added to the MM/SC aggregate or suspension. ### Claim 18 A method of generating an embryonic kidney organoid, the method comprising the steps of: providing a kidney mold device having a ureteric bud (UB) cell chamber and a metanephric mesenchymal (MM) cell chamber, wherein the MM cell chamber is fluidly connected to the UB cell chamber; inserting a plug into the kidney mold device MM cell chamber; seeding UB cells into the kidney mold device UB cell chamber to generate a UB tube; removing the plug by extraction or dissolution; generating a MM/somatic cell aggregate or suspension; transferring the MM/somatic cell aggregate or suspension into the kidney mold device MM cell chamber; and culturing the kidney mold device in growth media to generate an embryonic kidney organoid. ### Claim 19 (depends on 18) The method of claim 18, wherein the plug comprises a dissolvable material selected from the group consisting of gelatin, hydrogel, and polymer.