# US 10,117,968 B2 · Method of printing a tissue construct with embedded vasculature > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 10,117,968 B2 - Document ID: US10117968B2 - Status: Granted - Kind: B2 - Application number: 15/146,613 - Filed: 2014-11-04 - Published: 2016-10-06 - Granted: 2018-11-06 - Earliest claimed: 2013-11-05 - Assignees: President and Fellows of Harvard College - Applicants: President and Fellows of Harvard College - Inventors: Jennifer A. Lewis; David B. Kolesky; Mark A. Skylar-Scott; Kimberly A. Homan; Ryan L. Truby; Amelia Sydney Gladman - Web page: https://patentgrep.com/patent/US10117968B2 ## CPC - A61L 27/507: {for artificial blood vessels (apparatus for applying cells on a blood vessel prosthesis A61F2/062)} - A61F 2/06: Blood vessels - A61F 2/105: {Skin implants, e.g. artificial skin} - A61L 27/222: {Gelatin} - A61L 27/225: {Fibrin; Fibrinogen} - A61L 27/3633: {Extracellular matrix [ECM]} - A61L 27/3808: {Endothelial cells} - A61L 27/3813: {Epithelial cells, e.g. keratinocytes, urothelial cells} - A61L 27/3826: {Muscle cells, e.g. smooth muscle cells} - B29C 64/106: using only liquids or viscous materials, e.g. depositing a continuous bead of viscous material - B29C 64/118: using filamentary material being melted, e.g. fused deposition modelling [FDM] - B29C 64/40: Structures for supporting 3D objects during manufacture and intended to be sacrificed after completion thereof - B33Y 10/00: Processes of additive manufacturing - B33Y 70/00: Materials specially adapted for additive manufacturing - B33Y 80/00: Products made by additive manufacturing - A61F 2240/002: Designing or making customized prostheses - A61L 2430/34: for soft tissue reconstruction - 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)} - 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) - A61F: FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS (dental prosthetics A61C) - 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 - 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 A printed tissue construct comprises one or more tissue patterns, where each tissue pattern comprises a plurality of viable cells of one or more predetermined cell types. A network of vascular channels interpenetrates the one or more tissue patterns. An extracellular matrix composition at least partially surrounds the one or more tissue patterns and the network of vascular channels. A method of printing a tissue construct with embedded vasculature comprises depositing one or more cell-laden filaments, each comprising a plurality of viable cells, on a substrate to form one or more tissue patterns. Each of the one or more tissue patterns comprises one or more predetermined cell types. One or more sacrificial filaments, each comprising a fugitive ink, are deposited on the substrate to form a vascular pattern interpenetrating the one or more tissue patterns. The vascular pattern and the one or more tissue patterns are at least partially surrounded with an extracellular matrix composition. The fugitive ink is then removed to create vascular channels in the extracellular matrix composition, thereby forming an interpenetrating vascular network in a tissue construct. ## Claims ### Claim 1 A method of printing a tissue construct, comprising: depositing one or more cell-laden filaments each comprising a plurality of viable cells on a substrate to form one or more tissue patterns, each of the tissue patterns comprising one or more predetermined cell types; depositing one or more sacrificial filaments on the substrate to form a vascular channel pattern interpenetrating the one or more tissue patterns, each of the sacrificial filaments comprising a fugitive ink, wherein the fugitive ink comprises additional viable cells; at least partially surrounding the one or more tissue patterns and the vascular channel pattern with an extracellular matrix composition; and removing the fugitive ink to create an interconnected vascular channel network interpenetrating the one or more tissue patterns in the extracellular matrix composition, wherein at least a portion of the additional viable cells remains in the interconnected vascular channel network after removal of the fugitive ink. ### Claim 2 (depends on 1) The method of claim 1, wherein the additional viable cells are at least one of endothelial cells, smooth muscle cells, or connective tissue cells. ### Claim 3 (depends on 1) The method of claim 1, wherein each of the one or more cell-laden filaments further comprise an extracellular matrix material comprising one or more of gelatin, fibrin, and gelatin methacrylate. ### Claim 4 (depends on 1) The method of claim 1, wherein at least one of the one or more cell-laden filaments further comprises one or more functional chemical substances selected from the group consisting of: drugs, small molecules, toxins, proteins, and hormones. ### Claim 5 (depends on 1) The method of claim 1, wherein each of the one or more cell-laden filaments and each of the one or more sacrificial filaments are extruded from a nozzle at room temperature before being deposited on the substrate. ### Claim 6 (depends on 1) The method of claim 1, wherein removing the fugitive ink comprises cooling the one or more sacrificial filaments. ### Claim 7 (depends on 1) The method of claim 1, further comprising at least one of the following steps: after removing the fugitive ink, injecting a suspension of endothelial cells into the interconnected vascular channel network; or depositing one or more structural filaments layer by layer on the substrate to form a mold prior to at least partially surrounding the one or more tissue patterns and the vascular channel pattern with the extracellular matrix composition. ### Claim 8 (depends on 7) The method of claim 7, wherein the mold comprises flow channels in fluid communication with the vascular channels for perfusion thereof after removal of the fugitive ink. ### Claim 9 (depends on 7) The method of claim 7, further comprising: depositing one or more additional sacrificial filaments on the substrate to form a second vascular channel pattern, each of the additional sacrificial filaments comprising a second fugitive ink; at least partially surrounding the second vascular channel pattern with the extracellular matrix composition, and removing the second fugitive ink to create a second interconnected vascular channel network in the extracellular matrix composition, wherein, optionally, the second fugitive ink comprises additional viable cells, and wherein at least a portion of the additional viable cells remain in the second interconnected vascular channel network after removal of the second fugitive ink. ### Claim 10 (depends on 9) The method of claim 9, further comprising, after removing the second fugitive ink, injecting a suspension of additional viable cells into the second interconnected vascular channel network, wherein the additional viable cells comprise epithelial cells. ### Claim 11 (depends on 9) The method of claim 9, wherein the at least partial surrounding of the one or more tissue patterns and the vascular channel pattern with the extracellular matrix composition occurs during deposition of the one or more cell-laden filaments and the one or more sacrificial filaments, the one or more tissue patterns and the vascular channel pattern thereby being formed and embedded simultaneously in the extracellular matrix composition.