# US 10,119,107 B2 · Automated cell and tissue bioprinter > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 10,119,107 B2 - Document ID: US10119107B2 - Status: Granted - Kind: B2 - Application number: 14/890,998 - Filed: 2014-05-30 - Published: 2016-03-24 - Granted: 2018-11-06 - Earliest claimed: 2013-05-30 - Assignees: The University of Akron - Applicants: Hossein Tavana; David Petrak - Inventors: Hossein Tavana; David Petrak - Web page: https://patentgrep.com/patent/US10119107B2 ## CPC - C12M 21/08: {for producing artificial tissue or for ex-vivo cultivation of tissue (prostheses A61F2/00, grafts A61L27/00)} - B01L 3/0268: {using pulse dispensing or spraying, eg. inkjet type, piezo actuated ejection of droplets from capillaries} - B29C 64/112: using individual droplets, e.g. from jetting heads - B29C 64/393: for controlling or regulating additive manufacturing processes - B33Y 70/10: Composites of different types of material, e.g. mixtures of ceramics and polymers or mixtures of metals and biomaterials - B29K 2005/00: Use of polysaccharides or derivatives as moulding material - B29L 2031/7532: {Artificial members, protheses} - B33Y 10/00: Processes of additive manufacturing - B33Y 30/00: Apparatus for additive manufacturing; Details thereof or accessories therefor - B33Y 50/02: for controlling or regulating additive manufacturing processes - C12M 23/50: {Means for positioning or orientating the apparatus (C12M41/08 takes precedence)} - C12M 33/00: {Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus (chemical or physical laboratory apparatus in general B01L, devices for taking cell samples A61B10/0045, withdrawing or distributing predetermined quantities of fluid B01L99/00)} - 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} - B01L: CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE - B29C: SHAPING OR JOINING OF PLASTICS; SHAPING OF MATERIAL IN A PLASTIC STATE, NOT OTHERWISE PROVIDED FOR; AFTER-TREATMENT OF THE SHAPED PRODUCTS, e.g. REPAIRING (making preforms B29B11/00; making laminated products by combining previously unconnected layers which become one product whose layers will remain together B32B37/00 - B32B41/00) - B33Y: ADDITIVE MANUFACTURING, i.e. MANUFACTURING OF THREE-DIMENSIONAL [3D] OBJECTS BY ADDITIVE DEPOSITION, ADDITIVE AGGLOMERATION OR ADDITIVE LAYERING, e.g. BY 3D PRINTING, STEREOLITHOGRAPHY OR SELECTIVE LASER SINTERING - B29K: INDEXING SCHEME ASSOCIATED WITH SUBCLASSES B29B, B29C OR B29D, RELATING TO MOULDING MATERIALS OR TO MATERIALS FOR {MOULDS, } REINFORCEMENTS, FILLERS OR PREFORMED PARTS, e.g. INSERTS - B29L: INDEXING SCHEME ASSOCIATED WITH SUBCLASS B29C, RELATING TO PARTICULAR ARTICLES ## Abstract The present invention are directed to a novel automated cell bioprinter and related methods for making three-dimensional tissue constructs with spatial organization of cells that provides: (i) organized cell placement and spatial assembly of multiple cell types in a reproducible manner (ii) direct and non-contact assembly of multiple cell types and/or cell layers without exerting damaging forces on the cells or tissues, (iii) retention of cell viability and functionality during assembly steps and long periods of culture, (iv) minimal use of harmful and toxic chemicals such as profuse amounts of mineral oil and buffer reagent, and (v) efficient layer-to-layer assembly of cell layers within the three-dimensional construct while (vi) avoiding the use of complicated surface treatments that prevents scale-up, and (vii) avoiding mutation-inducing radiations such as UV used to photocrosslink hydrogels. ## Claims ### Claim 1 A method for the scaffold free bioprinting of cells comprising: A. providing an apparatus comprising a biomaterial surface upon which a plurality of cells are to be printed; one or more printing tips; a cartridge for holding said one or more printing tips; and a three-axis motion control system configured to move said cartridge in three dimensions with respect to said biomaterial surface; B. preparing an aqueous cell containing medium containing a first aqueous polymer; C. loading said aqueous cell containing medium into the one or more printing tips; D. preparing a second aqueous polymer solution, wherein said second aqueous polymer solution is less dense than and immiscible with said aqueous cell containing medium so that when the second aqueous polymer solution and said aqueous cell containing medium are mixed a partition is formed there between with the second aqueous polymer solution partitioned on the top and the aqueous cell containing medium on the bottom; E. preparing the biomaterial surface comprising a decellularized matrix upon which the cells contained in said aqueous cell containing medium are to be printed; F. covering said biomaterial surface with said second aqueous polymer solution to a depth of from about 1 to about 15 mm; G. inserting said one or more printing tips into openings in said cartridge configured to receive said printing tips; H. programming the three-axis motion control system to move said cartridge in three dimensions and at a predetermined speed to place the printing tips at specific predetermined locations as the aqueous cell containing medium is dispensed onto the biomaterial surface; and I. dispensing the aqueous cell containing medium onto the biomaterial surface at specific predetermined locations and at a predetermined speed. ### Claim 2 (depends on 1) The method of claim 1 wherein said first aqueous polymer is dextran. ### Claim 3 (depends on 1) The method of claim 1 wherein said second aqueous polymer solution contains polyethylene glycol (PEG). ### Claim 4 (depends on 1) The method of claim 1 wherein the interfacial tension between the second aqueous polymer solution and the aqueous cell containing medium is from about 10 to about 14 μN/m. ### Claim 5 (depends on 1) The method of claim 1 wherein and the interfacial tension between the second aqueous polymer solution and the aqueous cell containing medium is 12 μN/m. ### Claim 6 (depends on 1) The method of claim 1 wherein the cells are selected from the cell types consisting of MDA-MB-231 breast cancer cells, C2C12 mouse myoblast cells, embryonic stem cells, and combinations thereof. ### Claim 7 (depends on 1) The method of claim 1 wherein the aqueous cell containing medium is dispensed from said printing tips by the force of gravity. ### Claim 8 (depends on 1) The method of claim 1 wherein said biomaterial surface comprises a high-density decellularized matrix laid down by a 95-100% confluent cell monolayer of any cell type. ### Claim 9 (depends on 1) The method of claim 1 further comprising dispensing the aqueous cell containing medium onto the biomaterial surface in one or more linear patterns. ### Claim 10 (depends on 1) The method of claim 1 further comprising: I. allowing the cells in said aqueous cell containing medium to adhere to the biomaterial surface; J. rinsing any unadhered cells off of said biomaterial surface; K. covering said biomaterial surface and any cells adhered thereto with said second aqueous polymer solution to a depth of from about 1 to about 15 mm and L. repeating steps A through H to apply a second layer of aqueous cell containing medium upon said biomaterial surface. ### Claim 11 (depends on 10) The method of claim 10 further comprising: M. repeating steps A through L to apply additional cells upon said biomaterial surface and/or previously adhered cells to produce a three dimensional array of cells. ### Claim 12 (depends on 9) The method of claim 9 wherein said one or more linear patterns are reproducible. ### Claim 13 (depends on 9) The method of claim 9 wherein said one or more linear patterns are stable. ### Claim 14 (depends on 9) The method of claim 9 wherein each of said one or more linear patterns has a width of from about 254 μm to about 386 μm. ### Claim 15 (depends on 9) The method of claim 9 wherein each of said one or more linear patterns has a printing resolution of from 257.5 μm to about 386.5 μm. ### Claim 16 (depends on 9) The method of claim 9 wherein the Laplace Pressure is 130 mPa or less.