Granted Patent B2

US 12,630,795 · App. 17/284,976 · Granted May 19, 2026

Inventors: Goshi Kuno (Mie, JP); Yu Imaizumi (Mie, JP)

Assignee: TOSOH CORPORATION

C12M25/14C12M23/20C12N11/08C12N2539/10

Abstract

Claims (37)

1 . A cell culture substrate comprising:

a substrate, and

a stimulus-responsive polymer uniformly coated on the entire surface of the substrate,

wherein the stimulus-responsive polymer is a block copolymer having a water-insoluble block segment and a stimulus-responsive block segment,

wherein the cell culture substrate comprises the following two regions (A) and (B):

(A) an island-shaped region having cell proliferation properties and stimulus responsiveness and having an area of 0.001 to 5 mm 2 ; and

(B) a region adjacent to the region (A) and having no cell proliferation properties,

wherein the region (A) is composed of the following two regions (A1) and (A2), and the region (B) is composed of the following two regions (B1) and (B2):

(A1) cell proliferative region formed in the substrate;

(A2) a first region formed in the stimulus-responsive polymer;

(B1) substrate adhesion region formed in the substrate, in which the stimulus-responsive polymer coated on the substrate adheres to the substrate; and

(B2) second region formed in the stimulus-responsive polymer,

wherein the cell culture substrate having (A) the island-shaped region having cell proliferation properties and (B) the region adjacent to the region (A) and having no cell proliferation properties includes a layer containing the stimulus-responsive polymer formed on the substrate,

wherein the layer thickness of the layer containing the stimulus-responsive polymer is 20 nm or more and 50 nm or less,

wherein a ratio of an average roughness of the layer to a layer thickness of the layer is from 0.5 or more to 1 or less, and

wherein the regions (A1) are formed by a hydrophilization treatment, which is any of a plasma treatment, a UV treatment, and a corona treatment or a combination of a plurality thereof before coating the stimulus-responsive polymer on the substrate.

2 . The cell culture substrate according to claim 1 ,

wherein the region (A) is (i) formed by dispersing a temperature-responsive region having a diameter of 10 to 500 nm in a cell proliferative region, or (ii) formed by dispersing a cell proliferative region having a diameter of 10 to 500 nm in a temperature-responsive region having no cell proliferation properties.

3 . The cell culture substrate according to claim 1 ,

wherein the stimulus-responsive polymer is a block copolymer having (i) a water-insoluble block segment and (ii) a stimulus-responsive block segment exceeding 90 wt % of the block copolymer.

4 . The cell culture substrate according to claim 1 ,

wherein the region (A) is a plasma treatment region.

5 . The cell culture substrate according to claim 1 ,

wherein the cell culture substrate is used for spheroid formation for pluripotent stem cells.

6 . A method for producing the cell culture substrate according to claim 1 , comprising the following processes (1) and (2):

(1) a process of forming an island-shaped region having cell proliferation properties and an area of 0.001 to 5 mm 2 on a surface of a substrate having no cell proliferation properties; and

(2) a process of forming a layer of a stimulus-responsive substance on the surface of the substrate.

7 . The method for producing the cell culture substrate according to claim 6 ,

wherein, in the process (1), on the surface of the substrate having no cell proliferation properties, a region having cell proliferation properties is formed by any of a plasma treatment, a UV treatment, and a corona treatment or a combination of a plurality thereof.

8 . The method for producing the cell culture substrate according to claim 6 , wherein the substrate used in the process (1) has cell adhesion properties but has no cell proliferation properties.

9 . The method for producing the cell culture substrate according to claim 7 ,

wherein the process (1) comprises a process of attaching a surface protective film having holes with an area of 0.001 to 10 mm 2 to the substrate.

10 . A method for producing spheroids, comprising the following processes (i) to (iii):

(i) a process of seeding a cell on the cell culture substrate according to claim 1 ;

(ii) a process of culturing the seeded cells and forming a colony of cells adhered to the cell culture substrate; and

(iii) a process of separating at least a part of the colony from the cell culture substrate by applying an external stimulus and forming a cell spheroid.

11 . The cell culture substrate according to claim 1 , wherein the water-insoluble block segment is a polymer of at least one monomer selected from the group consisting of n-butyl acrylate, n-butyl methacrylate, isobutyl acrylate, isobutyl methacrylate, t-butyl acrylate, t-butyl methacrylate, n-hexyl acrylate, n-hexyl methacrylate, n-octyl acrylate, n-octyl methacrylate, n-decyl acrylate, n-decyl methacrylate, n-dodecyl acrylate, n-dodecyl methacrylate, n-tetradecyl acrylate, and n-tetradecyl methacrylate.

Assignments (1)

Priority Claims (1)

Continuity (1)

References Cited (42)