# US 2023/0056844 A1 · Cell culture substrate and manufacturing method therefor > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 2023/0056844 A1 - Document ID: US20230056844A1 - Status: Published application - Kind: A1 - Application number: 17/788,886 - Filed: 2020-12-28 - Published: 2023-02-23 - Earliest claimed: 2019-12-27 - Assignees: Amolifescience Co., Ltd. - Applicants: Amolifescience Co., Ltd. - Inventors: Hyeong Taek KIM; Dong Sik SEO; Hui Gwan GOO - Web page: https://patentgrep.com/patent/US20230056844A1 ## CPC - C12M 23/20: {Material Coatings (immunocoatings C12M25/00)} - C12N 5/0068: {General culture methods using substrates (for specific animal cell type C12N5/06)} - C07K 14/43504: {from invertebrates} - C12M 25/02: {Membranes; Filters (filters or filtration in general B01D24/00-B01D41/00)} - C12N 2533/50: Proteins - 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) - C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00) ## Abstract A cell culture substrate including a fibrous web in which fibers are integrated, and a cell culture coating layer comprising a coating film connecting at least some fibers from among fibers positioned on one surface of the fibrous web, wherein the cell culture coating layer is realized through a fusion protein for cell culture in which a functional peptide is bound to a mussel adhesive protein. Thus, the substrate can be stored at room temperature for a long period of time, i.e., several years, despite containing protein-like substances that aid in cell culture, and thus exhibits very high storage stability. At the same time, the activity of substances that aid in cell culture is maintained at the same level or is reduced only minimally, thus enabling cell culture at an initially designed level. ## Claims ### Claim 1 A cell culture substrate, comprising: a fibrous web in which fibers are integrated; and a cell culture coating layer comprising a coating film connecting at least some of the fibers positioned on one surface of the fibrous web, wherein the cell culture coating layer is formed with a fusion protein for cell culture in which a functional peptide is bound to a mussel adhesive protein. ### Claim 2 (depends on 1) The cell culture substrate according to claim 1, wherein the functional peptide has a function of promoting any one or more of adhesion, migration, proliferation and differentiation of a cell. ### Claim 3 (depends on 1) The cell culture substrate according to claim 1, wherein the fibrous web includes any one or more components selected from the group consisting of polystyrene (PS), polyester, polyethersulfone (PES), polyvinylidene fluoride (PVDF), polydimethylsiloxane (PDMS), polyamide, polyimide, polyethylene and polypropylene. ### Claim 4 (depends on 1) The cell culture substrate according to claim 1, wherein the mussel adhesive protein is any one protein selected from the group consisting of amino acid sequences of SEQ ID NO: 1 to SEQ ID NO: 14, or a protein to which one or more amino acid sequences selected from the group are linked. ### Claim 5 (depends on 1) The cell culture substrate according to claim 1, wherein the functional peptide comprises an RGD sequence. ### Claim 6 (depends on 1) The cell culture substrate according to claim 1, wherein the functional peptide is any one or more peptides selected from the group consisting of amino acid sequences of SEQ ID NO: 15 to SEQ ID NO: 19, or a peptide to which one or more amino acid sequences selected from the group are linked. ### Claim 7 (depends on 1) The cell culture substrate according to claim 1, further comprising a support disposed on the other surface opposite to the one surface of the fibrous web. ### Claim 8 (depends on 1) The cell culture substrate according to claim 1, wherein the fibrous web has an average diameter of 200 to 1000 nm, a thickness of 2 to 20 μm, and a basis weight of 3 to 20 g/m2. ### Claim 9 A method for manufacturing a cell culture substrate, comprising the steps of: (1) preparing an active solution containing a carbodiimide-based coupling agent and a reactive agent and a fusion protein for cell culture in which a functional peptide is bound to a mussel adhesive protein; (2) preparing a cell culture coating composition by mixing the prepared active solution and the prepared fusion protein for cell culture; and (3) forming a cell culture coating layer by treating the cell culture coating composition on a surface of a fibrous web. ### Claim 10 (depends on 9) The method for manufacturing a cell culture substrate according to claim 9, wherein the carbodiimide-based coupling agent is 1-ethyl-3-(3-dimethylaminopropyl carbodiimide hydrochloride (EDC) or N,N′-dicyclohexylcarboimide (DCC), and the reactive agent is N-hydroxysulfosuccinimide (Sulfo-NHS). ### Claim 11 (depends on 9) The method for manufacturing a cell culture substrate according to claim 9, wherein the carbodiimide-based coupling agent and the reactive agent are contained in the active solution in a weight ratio of 1:0.1 to 10, in the cell culture coating composition, the carbodiimide-based coupling agent is mixed in an amount of 1:1 to 100 parts by weight with respect to 100 parts by weight of the fusion protein for cell culture. ### Claim 12 A cell culture coating composition for a porous cell culture substrate which forms a coating layer that blocks at least some pores on a surface of the porous cell culture substrate, the cell culture coating composition comprising a fusion protein for cell culture in which a functional peptide is bound to a mussel adhesive protein, a carbodiimide-based coupling agent, and a reactive agent.