# US 11,078,459 B2 · Integrated methods for precision manufacturing of tissue engineering scaffolds > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 11,078,459 B2 - Document ID: US11078459B2 - Status: Granted - Kind: B2 - Application number: 15/998,685 - Filed: 2018-08-15 - Published: 2019-04-11 - Granted: 2021-08-03 - Earliest claimed: 2017-08-15 - Assignees: The Trustees of the Stevens Institute of Technology - Applicants: The Trustees of the Stevens Institute of Technology - Inventors: Filippos Tourlomousis; Robert Chang; Dilhan Kalyon - Web page: https://patentgrep.com/patent/US11078459B2 ## CPC - C12N 5/0068: {General culture methods using substrates (for specific animal cell type C12N5/06)} - A61K 35/28: Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells - A61L 27/3834: {Cells able to produce different cell types, e.g. hematopoietic stem cells, mesenchymal stem cells, marrow stromal cells, embryonic stem cells} - B33Y 10/00: Processes of additive manufacturing - B33Y 70/00: Materials specially adapted for additive manufacturing - C12N 5/0662: {Stem cells} - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - 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) - 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) - 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 ## Abstract Methods for the development and integration of multiple apparatuses and methods for achieving administration of stem cell therapies include precision manufacturing of tissue scaffolds and/or bioreactor substrates. The nano/microscale fiber material extrusion typifying the electrospinning process is married with the fiber alignment and layering characteristic of an additive manufacturing process. The method generates porous fibrous 3-D meshes with precision controlled structures from biopolymer melts and solutions and gels, blends, and suspensions with and without cells. A method of tracking the migration histories and shapes of stem cells on scaffold surfaces relies on immunofluorescent imaging and automated algorithms based on machine learning. The combination of the precision manufacturing method and the method of cell tracking and cell shape statistics, along with understanding of the intimate relationship between the cell shape/phenotype and scaffold architecture leads to an integrated method for cultivating and harvesting cells having desired phenotypes. ## Claims ### Claim 1 A three-dimensional scaffold, comprising a porous triangular microarchitecture having a 0-90 degree or 0-45 degree pore structure provided by a plurality of woven porous substrates stacked on each other, the porous microarchitecture having geometrical feature sizes of about 100 microns or smaller, and each of the woven porous substrates comprising a plurality of polycaprolactone fibers that are interwoven together. ### Claim 2 (depends on 1) The three-dimensional scaffold of claim 1, the porous triangular microarchitecture comprising a plurality of triangular pores that are substantially uniform in size and shape. ### Claim 3 (depends on 1) The three-dimensional scaffold of claim 1, the plurality of woven porous substrates being fabricated from a polycaprolactone melt using a melt electrowriting technique. ### Claim 4 (depends on 1) The three-dimensional scaffold of claim 1, the plurality of woven porous substrates being fabricated by melt electrowriting. ### Claim 5 (depends on 1) The three-dimensional scaffold of claim 1, the geometrical feature sizes being 10-100 μm. ### Claim 6 (depends on 1) The three-dimensional scaffold of claim 1, the plurality of fibers having a submicron diameter. ### Claim 7 (depends on 1) The three-dimensional scaffold of claim 1, the porous microarchitecture having a 0-45-135-90 degree pore structure. ### Claim 8 (depends on 1) A method of expanding cells in vitro, comprising: providing the three-dimensional scaffold of claim 1, seeding a plurality of cells on the three-dimensional scaffold, culturing the cells to obtain expanded cells that are substantially uniform in shape and phenotype. ### Claim 9 (depends on 8) The method of claim 8, wherein the cells are stem cells and the expanded cells maintain stemness. ### Claim 10 (depends on 8) The method of claim 8, wherein the expanded cells are homogenous. ### Claim 11 (depends on 8) The method of claim 8, further comprising determining a relationship between the porous microarchitecture and the shape and phenotype of the expanded cells. ### Claim 12 (depends on 8) The method of claim 8, further comprising generating a metrology framework that models and classifies cell confinement states for various porous microarchitectures. ### Claim 13 (depends on 8) The method of claim 8, further comprising classifying cell shapes and/or focal adhesions using machine learning. ### Claim 14 (depends on 13) The method of claim 13, wherein the machine learning is carried out using immunofluorescent imaging of the expanded cells. ### Claim 15 (depends on 13) The method of claim 13, wherein the immunofluorescent imaging comprises immunofluorescent labeling of positive and/or negative cell surface markers. ### Claim 16 (depends on 8) The method of claim 8, further comprising tuning so as to reproducibly harvesting targeted cell populations.