Cell culture substrate and manufacturing method therefor

Patent metadata

Field	Value
Publication number	US-12534703-B2
Application number	US-202017788886-A
Country	US
Kind code	B2
Filing date	Dec 28, 2020
Priority date	Dec 27, 2019
Publication date	Jan 27, 2026
Grant date	Jan 27, 2026

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.

First claim

The invention claimed is:

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 and blocking some pores formed on the surface, wherein the cell culture coating layer is formed with a fusion protein for cell culture in which a functional peptide that promotes any one or more of adhesion, migration, proliferation and differentiation of a cell is bound to a mussel adhesive protein.

2 . 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.

3 . 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.

4 . The cell culture substrate according to claim 1 , wherein the functional peptide comprises an RGD sequence.

5 . 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.

6 . 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.

7 . 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/m 2 .

8 . 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 comprising N-hydroxysulfosuccinimide (Sulfo-NHS), and preparing a fusion protein for cell culture in which a functional peptide that promotes any one or more of adhesion, migration, proliferation, and differentiation of a cell 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.

9 . The method for manufacturing a cell culture substrate according to claim 8 , wherein the carbodiimide-based coupling agent is 1-ethyl-3-(3-dimethylaminopropyl carbodiimide hydrochloride (EDC) or N,N′-dicyclohexylcarboimide (DCC).

10 . The method for manufacturing a cell culture substrate according to claim 8 , 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, and
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.

11 . 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 that promotes any one or more of adhesion, migration, proliferation, and differentiation of a cell is bound to a mussel adhesive protein, a carbodiimide-based coupling agent, and a reactive agent comprising N-hydroxysulfosuccinimide (Sulfo-NHS).

Assignees

• Amolifescience Co Ltd

Inventors

• Kim Hyeong Taek
• Seo Dong Sik
• Goo Hui Gwan

Classifications

• C12N2533/50

  Proteins · CPC title

• C12N5/0068 Primary

  General culture methods using substrates (for specific animal cell type C12N5/06) · CPC title

• C12M25/06

  Plates; Walls; Drawers; Multilayer plates · CPC title

• C07K14/43504

  from invertebrates · CPC title

• C12M25/02

  Membranes; Filters (filters or filtration in general B01D24/00-B01D41/00) · CPC title