Abstract: A method for manufacturing a cell mass adherently cultured on a cell culture substrate, in which the cell culture substrate comprises two regions (A) and (B), and the method comprising steps (1) to (3): (A) an island-shaped region having a cell proliferation property and an area of 0.001 to 1 mm2; and (B) a region adjacent to the region (A) and having no cell proliferation properties, (1) a step of preparing a cell suspension comprising a mesenchymal cell and an ectoderm-derived cell; (2) a step of bringing the cell suspension into contact with the cell culture substrate, and randomly adhering the two types of cells to the region (A), and in which, on the contact surface between the cell suspension and the cell culture substrate, a total number of cells of the two types of cells is 100 to 100,000 cells/cm2; and (3) a step of culturing the cell adhered to the region (A) and forming a cell mass comprising the two types of cells.

Abstract: There are provided a cell culture substrate that enables efficient spheroid formation for cells and can form spheroids having a uniform size and an arbitrary shape at a high cell viability, a method for producing the cell culture substrate, and a method of producing spheroids in which the cell culture substrate is used and a cell viability inside of the spheroids is excellent. A cell culture substrate includes a substrate and a stimulus-responsive polymer coated on the substrate, wherein the stimulus-responsive polymer is a block copolymer having a water-insoluble block segment and a stimulus-responsive block segment, and the cell culture substrate includes 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.