Cell culture substrate, method for producing cell culture substrate, and method for producing spheroids

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 mm2;
• 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.