Temperature-responsive cell culture substrate and method for producing same

Patent metadata

Field	Value
Publication number	US-9951308-B2
Application number	US-201415106470-A
Country	US
Kind code	B2
Filing date	Dec 11, 2014
Priority date	Dec 20, 2013
Publication date	Apr 24, 2018
Grant date	Apr 24, 2018

Abstract

Provided is a cell culture substrate, in which polymer (B) having a lower critical solution temperature contained in the substrate is a copolymer of a monomer (a) that becomes a hydrophobic polymer in homopolymerization and a monomer (b, c or d) that becomes a hydrophilic polymer in homopolymerization, which is uncrosslinked, and the lower critical solution temperature of the obtained copolymer (B) can be controlled widely by the types and ratio of the two monomers, to easily detach the cultured cells from the culture substrate surface rapidly without using protein hydrolase and collect the cells without damage. This cell culture substrate contains a polymer (A) of a (meth)acrylic acid ester monomer (a), one or more types of inorganic materials (C) selected from a water-swellable clay mineral and silica, and a polymer (B) having a lower critical solution temperature and including a monomer (a) and a monomer (b, c or d).

First claim

The invention claimed is:

1. A cell culture substrate, comprising:
a polymer (A) of a monomer (a) represented by the following Formula (1);
a polymer (B) having a lower critical solution temperature; and
one or more inorganic materials (C) selected from a water-swellable clay mineral and silica,
wherein the mass ratio ((C)/(A)) of the polymer (A) and the inorganic material (C) is in a range of 0.01 to 3;
wherein the polymer (B) is a copolymer (B1) of the monomer (a) 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 the monomer (a) and a polyethylene glycol chain-containing monomer (d) represented by the following Formula (3); and
wherein the content rate of the polymer (B) is in a range of 0.1% by mass to 40% by mass with respect to the entire cell culture substrate,

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

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

wherein n represents an integer of 2 to 20.

2. The cell culture substrate according to claim 1 ,
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.

3. The cell culture substrate according to claim 2 ,
wherein the water-swellable clay mineral is one or more clay minerals selected from water-swellable hectorite, water-swellable montmorillonite, water-swellable saponite, and water-swellable synthetic mica, which cause delamination in an aqueous medium (W) to provide 1 to 10 layers, and
wherein the silica is water dispersible colloidal silica.

4. A production method for the cell culture substrate according to claims 2 , the method comprising:
a first step of producing a dispersion liquid (L) of a complex (X) of the polymer (A) and the inorganic material (C) by mixing the monomer (a), the inorganic material (C), and a polymerization initiator (D) into a aqueous medium (W) such that the concentration of the inorganic material (C) in the aqueous medium (W) is in a range defined by the following Expression (4) or Expression (5), and then polymerizing the monomers (a); and
a second step of adding and mixing the polymer (B) into the dispersion liquid (L), and coating a support with the resulting mixture, followed by drying, in this order,
when Ra<0.19 is established, the concentration (% by mass) of the inorganic material (C)<12.4Ra+0.05 is satisfied; and  Expression (4)

when Ra≥0.19 is established, the concentration (% by mass) of the inorganic material (c)<0.87Ra+2.17 is satisfied  Expression (5),

wherein the concentration (% by mass) of the inorganic material (C) is a numerical value obtained by dividing the mass of the inorganic material (C) by the total mass of the aqueous medium (W) and the inorganic material (C) and then multiplying the obtained value by 100, and Ra represents the mass ratio ((C)/(A)) of the inorganic material (C) and the polymer (A).

5. The cell culture substrate according to claim 1 , wherein the water-swellable clay mineral is one or more clay minerals selected from water-swellable hectorite, water-swellable montmorillonite, water-swellable saponite, and water-swellable synthetic mica, which cause delamination in an aqueous medium (W) to provide 1 to 10 layers, and
wherein the silica is water dispersible colloidal silica.

6. A production method for the cell culture substrate according to claim 5 , the method comprising:
a first step of producing a dispersion liquid (L) of a complex (X) of the polymer (A) and the inorganic material (C) by mixing the monomer (a), the inorganic material (C), and a polymerization initiator (D) into a aqueous medium (W) such that the concentration of the inorganic material (C) in the aqueous medium (W) is in a range defined by the following Expression (4) or Expression (5), and then polymerizing the monomers (a); and
a second step of adding and mixing the polymer (B) into the dispersion liquid (L), and coating a support with the resulting mixture, followed by drying, in this order,
when Ra<0.19 is established, the concentration (% by mass) of the inorganic material (C)<12.4Ra+0.05 is satisfied; and  Expression (4);

when Ra≥0.19 is established, the concentration (% by mass) of the inorganic material (c)<0.87Ra+2.17 is satisfied   Expression (5):,

wherein the concentration (% by mass) of the inorganic material (C) is a numerical value obtained by dividing the mass of the inorganic material (C) by the total mass of the aqueous medium (W) and the inorganic material (C) and then multiplying the obtained value by 100, and Ra represents the mass ratio ((C)/(A)) of the inorganic material (C) and the polymer (A).

7. A production method for the cell culture substrate according to claim 1 , the method comprising:
a first step of producing a dispersion liquid (L) of a complex (X) of the polymer (A) and the inorganic material (C) by mixing the monomer (a), the inorganic material (C), and a polymerization initiator (D) into a aqueous medium (W) such that the concentration of the inorganic material (C) in the aqueous medium (W) is in a range defined by the following Expression (4) or Expression (5), and then polymerizing the monomers (a); and
a second step of adding and mixing the polymer (B) into the dispersion liquid (L), and coating a support with the resulting mixture, followed by drying, in this order,
when Ra<0.19 is established, the concentration (% by mass) of the inorganic material (C)<12.4Ra+0.05 is satisfied; and  Expression (4);

when Ra ≥0.19 is established, the concentration (% by mass) of the inorganic material (c)<0.87Ra+2.17 is satisfied  Expression (5),

wherein the concentration (% by mass) of the inorganic material (C) is a numerical value obtained by dividing the mass of the inorganic material (C) by the total mass of the aqueous medium (W) and the inorganic material (C) and then multiplying the obtained value by 100, and Ra represents the mass ratio ((C)/(A)) of the inorganic material (C) and the polymer (A).

8. A production method for the cell culture substrate according to claim 3 , the method comprising:
a first step of producing a dispersion liquid (L) of a complex (X) of the polymer (A) and the inorganic material (C) by mixing the monomer (a), the inorganic material (C), and a polymerization initiator (D) into a aqueous medium (W) such that the concentration of the inorganic material (C) in the aqueous medium (W) is in a range defined by the following Expression (4) or Expression (5), and then polymerizing the monomers (a); and
a second step of adding and mixing the polymer (B) into the dispersion liquid (L), and coating a support with the resulting mixture, followed by drying, in this order,
when Ra<0.19 is established, the concentration (% by mass) of the inorganic material (C)<12.4Ra+0.05 is satisfied; and  Expression (4);

when Ra≥0.19 is established, the concentration (% by mass) of the inorganic material (c)<0.87Ra+2.17 is satisfied  Expression (5),

wherein the concentration (% by mass) of the inorganic material (C) is a numerical value

Assignees

• Dainippon Ink & Chemicals

Inventors

• Takada Tetsuo

Classifications

• C08L33/14

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

• C12M25/14

  Scaffolds; Matrices (in general C12N5/0068) · CPC title

• C12M25/02

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

• C12N2539/10

  Coating allowing for selective detachment of cells, e.g. thermoreactive coating · CPC title

• C08L33/24

  Homopolymers or copolymers of amides or imides · CPC title