Three-dimensional reconstructed organoids in vitro are valuable for not only regenerative medicine but also drug development. However, the manipulation of conventional three-dimensional cultures is not simple. We describe a nylon membrane ring-embedded or a pressed silk sheet-embedded scaffold made of collagen "vitrigel" that can facilitate three-dimensional cultures for reconstructing an epithelial-mesenchymal model or a hard connective tissue model, respectively. Here we define vitrigel as a gel in a stable state produced by rehydration after the vitrification of a traditional hydrogel. The collagen vitrigel was successfully prepared in three steps involving a gelation process in which a cold and clear neutral salt solution containing type I collagen formed an opaque and soft gel by incubation at 37°C, a vitrification process in which the gel becomes a rigid material like glass after sufficient drying out, and finally a rehydration process to convert the vitrified material into a thin and transparent gel membrane with enhanced gel strength. The frameworkembedded collagen vitrigel scaffold that can be easily reversed by forceps was prepared by inserting a nylon ring or a silk sheet in the collagen solution prior to the gelation. The scaffold enabled culturing anchoragedependent cells on both surfaces of the collagen vitrigel by the manipulation of two-dimensional cultures and consequently resulted in reconstructing a three-dimensional organoid. An intestinal epithelial-mesenchymal model was reconstructed by coculturing fibroblasts on the opposite side of monolayered Caco-2 cells on the nylon ring-embedded collagen vitrigel. Also, fibroblasts seeded on both surfaces of the silk sheetembedded collagen vitrigel proliferated well and formed multilayers and some cells invaded into the vitrigel framed by the network of numerous strong silk filaments, suggesting a reconstruction of a hard connective tissue model. These data demonstrate that the collagen vitrigel is a valuable scaffold for tissue engineering.Key words: Scaffold; Tissue engineering; Collagen; Vitrigel; Three-dimensional culture; Organoid INTRODUCTIONincorporated culture systems utilizing scaffolds such as acellular dermis, small intestinal submucosa, and tissue/ Various three-dimensional culture systems have been organ sections for histopathology (TOSHI) (2,10,20,36); developed by devising the structures and components of and 5) premolded biodegradable polymer-incorporated cellular scaffolds to reconstruct organoids that can be culture systems utilizing a polyglycolic acid (PGA) scafutilized for in vitro normal or pathological models to fold (26,27). examine drug effects and/or toxicities, for ex vivo extraThese three-dimensional culture systems have concorporeal devices to assist defective organs, or for in tributed not only to basic life science but also to applied vivo grafts (37). Culture systems to reorganize a threebiomedical research. Human umbilical vein endothelial dimensional multicellular mass are classified into the folcells (HUVECs) culture...

A pressed silk sheet is a new biomaterial composed of a network of numerous cocoon filaments and having excellent mechanical strength and shape stability compared to a cotton-gauze sheet. To reconstruct a hard connective tissue using the silk sheet and type-I collagen as the scaffold for fibroblasts, three different three-dimensional floating culture systems were designed. "On sheet" system: fibroblasts were seeded on the silk sheet coated with collagen and the cell-attached sheet was cultured. "In gel" system: fibroblasts and the silk sheet were co-embedded in a collagen gel and the gel was cultured. "On vitrigel" system: fibroblasts were seeded on both sides of a collagen vitrigel involving the silk sheet and the vitrigel was cultured. The fibroblasts in all culture systems grew and formed disk-shaped connective tissue models involving the silk sheet by 14 days of culture. The "on sheet" and "on vitrigel" models retained a maximum elastic load of about 23 kgf and an ultimate tensile load of about 3.6 kgf, which were almost the same as for the individual silk sheet. However, the "in gel" system showed a low value for the tensile load. Cell damage following application of mechanical stress was lowest in the "on vitrigel" system. These data demonstrated the advantage of the "on vitrigel" system in reconstructing hard connective tissues. Such a novel culture method would contribute to a regenerative medicine for the failure of ligaments, tendons, and other connective tissues.