Three-dimensional (3D) cell spheroids have a demonstrated value for in vitro biological research and therapeutics development. Attempts to this technique focus on the development of effective methods for fabricating cell spheroids. Here, inspired by the heterogeneously textured wettability bumps (with hydrophilic peaks and hydrophobic bases) of Stenocara beetle, we present a biotemplated substrate with wettable hydrogel arrays for culturing the cell spheroids. The biotemplates were Morpho butterfly wings with chitin and protein components, which could provide a natural superhydrophobic surface without any modification. The droplet microarrays could be formed for cell spheroid culture on this bioinspired wing substrate by using the hydrogel patterns to hanging droplets. The hanging drop culture method on hydrogel-covered wings has the advantages of high speed, uniform size, and controllable diameter for the formation of 3D cell spheroids. It was demonstrated that drugs produced distinct responses in the 3D cell spheroids compared to conventional two-dimensional cell cultures. As the presented system does not require complex instruments and chemical modifications, our method can simply construct the desired wettability substrates with high biocompatibility for cell culture, drug testing, and other biomedical applications.
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