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BACKGROUND Three-dimensional (3D) cell-culture scaffolds are ideal in vitro models to bridge the gap between two-dimensional cell culture in vitro and in vivo cancer models. Construction of 3D scaffolds using two kinds of biomaterials has been reported, but there are still many defects. To improve the performance of the scaffolds for 3D cell culture of colonic carcinoma (CC) cells in vitro, we attempted to construct triple composite scaffolds using silk fibroin (SF), chitosan (Cs), and alginate (Alg). MATERIAL AND METHODS We explored the suitability of triple composite scaffolds of SF/Cs/Alg at ratios of 1: 1: 0.5, 1: 1: 1, and 1: 1: 2 for 3D culture of CC cells, and used the dual composite scaffold of SF/Cs (1: 1) as a control group. We analyzed the physicochemical characteristics of these scaffolds and studied cell adhesion, cell proliferation, migration, colony-forming ability, microstructure and ultrastructure, and spheroid-forming capacity of the commercially available CC cell line HCT-116 on the prepared scaffolds. RESULTS Our results show that SF/Cs/Alg (1: 1: 1) scaffolds demonstrated the best profile, the highest uniform porosity and connectivity, and excellent hydroscopicity, and also exhibited appropriate and controlled swelling and degradation characteristics. The adhesion, proliferation, colony-forming, and wound-healing assays, green fluorescent protein-labeled HCT116 cell imaging, 4',6-diamidino-2-phenylindole and DY-554-phalloidin staining, scanning electron microscopy, and haematoxylin and eosin staining revealed that the triple composite scaffolds of SF/CS/Alg (1: 1: 1) supported cell adhesion, proliferation, migration, colony-forming ability, and spheroid formation far better than the dual composite scaffold of SF/CS (1: 1). CONCLUSIONS This study successfully demonstrated the potential of SF/Cs/Alg (1: 1: 1) scaffold as an alternative for the 3D in vitro culture of CC cells.