Cell culture substrate

Patent metadata

Field	Value
Publication number	US-11499136-B2
Application number	US-201716467535-A
Country	US
Kind code	B2
Filing date	Dec 12, 2017
Priority date	Dec 22, 2016
Publication date	Nov 15, 2022
Grant date	Nov 15, 2022

Abstract

The present invention provides a cell culture substrate including a polymer having a lower critical solution temperature, the substrate including one or more inorganic materials selected from a water-swellable clay mineral and silica and further including an adhesive matrix in the substrate, in which the adhesive matrix is an extracellular matrix and/or an adhesive synthetic matrix. Furthermore, the invention is to provide a cell culture substrate in which the extracellular matrix is at least one selected from laminin, fibronectin, vitronectin, cadherin, and fragments thereof, and/or the adhesive synthetic matrix is poly[2-(methacryloyloxy)ethyl dimethyl-(3-sulfopropyl) ammonium hydroxide] or an oligopeptide-supporting polymer.

First claim

The invention claimed is:

1. A stem cell culture substrate comprising: a polymer having a lower critical solution temperature; one or more inorganic materials selected from a water-swellable clay mineral and silica; and an adhesive matrix, wherein the adhesive matrix is an extracellular matrix and/or an adhesive synthetic matrix,
wherein the extracellular matrix is at least one selected from laminin, fibronectin, vitronectin, cadherin, and fragments thereof, and
wherein the stem cell culture substrate has an average film thickness of 1,000 nm or less.

2. The stem cell culture substrate according to claim 1 , wherein the adhesive synthetic matrix is poly[2-(methacryloyloxy)ethyl dimethyl-(3-sulfopropyl) ammonium hydroxide] or an oligopeptide-supporting polymer.

3. The stem culture substrate according to claim 1 , wherein the polymer having a lower critical solution temperature is a polymer obtainable by polymerizing a monomer that gives a homopolymer having a lower critical solution temperature, and the monomer that gives a homopolymer having a lower critical solution temperature is at least one selected from N-isopropyl (meth)acrylamide, N-n-propyl (meth)acrylamide, N-cyclopropyl (meth)acrylamide, N-ethoxyethyl (meth)acrylamide, N-tetrahydrofurfuryl (meth)acrylamide, N-ethylacrylamide, N-ethyl-N-methylacrylamide, N,N-diethylacrylamide, N-methyl-N-n-propylacrylamide, N-methyl-N-isopropylacrylamide, N-acryloylpiperidine, and N-acryloylpyrrolidine.

4. The stem cell culture substrate according to claim 1 , wherein
the polymer having a lower critical solution temperature is at least one of
a copolymer (B1) of a monomer (a) represented by the following Formula (1) and a hydrophilic amide-based vinyl monomer (b),
a copolymer (B2) of the monomer (a) and a monomer (c) represented by the following Formula (2), or a copolymer (B3) of monomer (a) and a polyethylene glycol chain-containing monomer (d) represented by the following Formula (3), and
the percentage content of the polymer having a lower critical solution temperature with respect to the total amount of the cell culture substrate is 5% by mass to 99% by mass:

wherein R 1 represents a hydrogen atom or a methyl group; R 2 represents an alkylene group having 2 or 3 carbon atoms; and R 3 represents an alkyl group having 1 or 2 carbon atoms,

wherein R 4 represents a hydrogen atom or a methyl group; and R 5 represents an alkylene group having 2 or 3 carbon atoms, and

wherein n represents an integer of 2 to 20.

5. The stem culture substrate according to claim 4 , wherein the hydrophilic amide-based vinyl monomer (b) is at least one monomer selected from the group consisting of an N-substituted (meth)acrylamide derivative, an N,N-disubstituted (meth)acrylamide derivative, and N-vinylpyrrolidone.

6. The stem cell culture substrate according to claim 1 , further comprising a polymer (A) of the monomer (a) represented by the following Formula (1)

wherein R 1 represents a hydrogen atom or a methyl group; R 2 represents an alkylene group having 2 or 3 carbon atoms; and R 3 represents an alkyl group having 1 or 2 carbon atoms.

7. The stem culture substrate according to claim 1 , wherein the water-swellable clay mineral is one or more clay minerals that delaminate into 1 to 10 layers in an aqueous medium (W), the water-swellable clay mineral being selected from water-swellable hectorite, water-swellable montmorillonite, water-swellable saponite, and water-swellable synthetic mica, and the silica is water-dispersible colloidal silica.

8. The stem cell culture substrate according to claim 1 , wherein the cell culture substrate comprising a polymer having a lower critical solution temperature further includes thereon at least one protein selected from gelatin, collagen, and/or albumin.

9. The stem cell culture substrate according to claim 8 , wherein a polymer having a lower critical solution temperature; at least one protein selected from gelatin, collagen, and/or albumin; and an adhesive matrix are laminated in sequence.

10. The stem cell culture substrate according to claim 1 , wherein the cell culture substrate is a dry cell culture substrate.

11. The stem cell culture substrate according to claim 1 , wherein the cell culture substrate is laminated on a supporting medium.

12. A method for producing a dry stem cell culture substrate, the method comprising:
a step of coating a solution including at least one protein selected from gelatin, collagen, and/or albumin on a polymer having a lower critical solution temperature;
a step of further coating a solution including the adhesive matrix according to claim 1 to obtain a cell culture substrate; and
a step of drying the obtained stem cell culture substrate.

13. A cell culture equipment, comprising a supporting medium and the stem cell culture substrate according to claim 1 .

Assignees

• Fujifilm Corp
• Dainippon Ink & Chemicals

Inventors

• Takada Tetsuo
• Sakurai Naoto
• Isshiki Ayako
• SANTOU Noriko

Classifications

• C08L33/14

  of esters containing halogen, nitrogen, sulfur, or oxygen atoms in addition to the carboxy oxygen · CPC title

• C12N5/0068 Primary

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

• C01B33/44

  Products obtained from layered base-exchange silicates by ion-exchange with organic compounds such as ammonium, phosphonium or sulfonium compounds or by intercalation of organic compounds, e.g. organoclay material · CPC title

• C12M23/20 Primary

  Material Coatings (immunocoatings C12M25/00) · CPC title

• C12N2533/32

  Polylysine, polyornithine · CPC title