# US 10,368,980 B2 · Middle ear mucosa-like cell sheet, process of producing the same and method of using the same > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 10,368,980 B2 - Document ID: US10368980B2 - Status: Granted - Kind: B2 - Application number: 15/098,604 - Filed: 2016-04-14 - Published: 2016-08-04 - Granted: 2019-08-06 - Earliest claimed: 2010-09-15 - Assignees: Tokyo Women's Medical University; The Jikei University; Cellseed Inc. - Applicants: Tokyo Women's Medical University; The Jikei University; CellSeed Inc. - Inventors: Takanori Hama; Hiromi Kojima; Hiroshi Moriyama; Masayuki Yamato; Teruo Okano - Web page: https://patentgrep.com/patent/US10368980B2 ## CPC - A61F 2/18: Internal ear or nose parts, e.g. ear-drums {(implants for middle ear ventilation or drainage A61F11/202)} - A61L 27/3804: {characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells} - A61L 27/3813: {Epithelial cells, e.g. keratinocytes, urothelial cells} - A61L 27/3839: {characterised by the site of application in the body (materials for artificial blood vessels A61L27/507; materials for use in artificial skin A61L27/60)} - A61L 27/3886: {comprising two or more cell types} - A61P 27/16: Otologicals - C12N 5/062: {Sensory transducers, e.g. photoreceptors; Sensory neurons, e.g. for hearing, taste, smell, pH, touch, temperature, pain} - A61F 2002/183: {Ear parts} - A61F 2310/00365: Proteins; Polypeptides; Degradation products thereof - A61K 35/12: Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells (vaccines or medicinal preparations containing antigens or antibodies A61K39/00) - A61L 2430/14: for ear reconstruction or ear implants, e.g. implantable hearing aids - C12N 2533/30: Synthetic polymers - 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) - 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) - A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS - 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) ## Abstract The purpose of the present invention is to obtain an alternative to a substitute of the mucosa in the middle ear which is engrafted on the surface of the bone in the middle ear, hyperplasia of the granulation tissue and the bone and the development of the fibroblast cells in the middle ear are suppressed, and to obtain a middle ear mucosa-like cell sheet retaining cilia in the surface layer, comprising culturing nasal epithelium cells on a cell culture substrate coated with a polymer whose hydration force changes within a temperature range of 0 to 80° C., wherein the cells are cultured within a temperature range where the hydration force of the polymer is weak, and then changing the temperature to a temperature at which the hydration force is strong to recover the cultured cell sheet. ## Claims ### Claim 1 A method of transplanting a middle ear mucosa-like cell sheet into a subject in need thereof, the method comprising (i) preparing a middle ear mucosa-like cell sheet containing nasal epithelium cells retaining cilia thereof and nasal mucosa cells, the nasal epithelium cells originating from inferior turbinate mucosa, middle turbinate mucosa, or mucosa in paranasal sinus, wherein the nasal epithelium cells in the middle ear mucosa-like cell sheet comprise flat forms of the nasal epithelium cells, said preparing including: a) detaching a portion of nasal mucosa epithelial tissue from inferior turbinate mucosa, middle turbinate mucosa, or mucosa in paranasal sinus while excluding gland cells; b) explanting the portion of nasal mucosa epithelial tissue on a cell culture substrate to cause proliferation of mucosal cells from the portion of nasal mucosa epithelial tissue, wherein the cell culture substrate is coated with a cell-adhering protein; c) culturing the proliferated mucosal cells on a cell insert coated with a polymer which exhibits a weaker hydration force in a first temperature range than in a second temperature range, said culturing being carried out within the first temperature range such that the cultured middle ear mucosa-like cells form a sheet on the cell insert; d) changing the temperature from within the first temperature range to the second temperature range to thereby detach the sheet of cultured middle ear mucosa-like cells from the cell insert while retaining the form of the sheet; and (ii) transplanting the middle ear mucosa-like cell sheet to a middle ear cavity or a mastoid antrum of a subject. ### Claim 2 (depends on 1) The method of claim 1, wherein the middle ear mucosa-like cell sheet is transplanted to a transplantation site preliminarily coated with a fibrin glue. ### Claim 3 (depends on 1) The method of claim 1, wherein the subject is in need of treatment for cholesteatoma of middle ear, adhesive otitis, postoperative mucocele of paranasal sinus, frontal sinus cyst, oropharyngeal cancer, oropharyngeal cancer, paranasal cancer or laryngeal cancer, or reconstruction of a middle ear cavity. ### Claim 4 (depends on 1) The method of claim 1, wherein the detached the middle ear mucosa-like cell sheet comprises an adhesive protein that is intact and was not damaged by a protease. ### Claim 5 (depends on 1) The method of claim 1, wherein the middle ear mucosa-like cell sheet is multilayered. ### Claim 6 (depends on 1) The method of claim 1, wherein the middle ear mucosa-like cell sheet has a ventilatory function. ### Claim 7 (depends on 1) The method of claim 1, wherein the middle ear mucosa-like cell sheet has a granulation tissue inhibitory function. ### Claim 8 (depends on 1) The method of claim 1, wherein the middle ear mucosa-like cell sheet has a mucin secretion function. ### Claim 9 (depends on 1) The method of claim 1, wherein the middle ear mucosa-like cell sheet is transgenic. ### Claim 10 (depends on 1) The method of claim 1, wherein the middle ear mucosa-like cell sheet further comprises one or more types of cells selected from the group consisting of vascular endothelial cells, an endothelial precursor cell, fat-derived cells, and mesenchymal stem cells. ### Claim 11 (depends on 1) The method of claim 1, wherein the middle ear mucosa-like cell sheet further comprises two or more middle ear mucosa-like cell layers, wherein each of the middle ear mucosa-like cell layers comprises one or more types of cells selected from the group consisting of vascular endothelial cells, an endothelial precursor cells, fat-derived cells, and mesenchymal stem cells. ### Claim 12 A method of preparing a middle ear mucosa-like cell sheet containing nasal epithelium cells to be transplanted into a subject in need thereof, the middle ear mucosa-like cell sheet containing nasal epithelium cells retaining cilia thereof and nasal mucosa cells, the nasal epithelium cells originating from inferior turbinate mucosa, middle turbinate mucosa, or mucosa in paranasal sinus, wherein the nasal epithelium cells in the middle ear mucosa-like cell sheet comprise flat forms of the nasal epithelium cells, said method comprising: a) detaching a portion of nasal mucosa epithelial tissue from inferior turbinate mucosa, middle turbinate mucosa, or mucosa in paranasal sinus while excluding gland cells, b) explanting the portion of nasal mucosa epithelial tissue on a cell culture substrate to cause proliferation of mucosal cells from the portion of nasal mucosa epithelial tissue, wherein the cell culture substrate is coated with a cell-adhering protein, c) culturing the proliferated mucosal cells on a cell insert coated with a polymer which exhibits a weaker hydration force in a first temperature range than in a second temperature range, said culturing being carried out within the first temperature range such that the cultured middle ear mucosa-like cells form a sheet on the cell insert, and d) changing the temperature from within the first temperature range to the second temperature range to thereby detach the sheet of cultured middle ear mucosa-like cells from the cell insert while retaining the form of the sheet. ### Claim 13 (depends on 1) The method of claim 1, wherein the cell-adhering protein is one or more cell-adhering protein(s) selected from a group consisting of collagen, laminin, laminin 5, fibronectin and matrigel. ### Claim 14 (depends on 1) The method of claim 1, wherein the cell-adhering protein is collagen. ### Claim 15 (depends on 12) The method of claim 12, wherein the cell-adhering protein is one or more cell-adhering protein(s) selected from a group consisting of collagen, laminin, laminin 5, fibronectin and matrigel. ### Claim 16 (depends on 12) The method of claim 12, wherein the cell-adhering protein is collagen.