Collagen Vitrigel: A Novel Scaffold that can Facilitate a Three-Dimensional Culture for Reconstructing Organoids

• Published in: Cell transplantation Vol. 13; no. 4; pp. 463 - 474
• Main Authors: Takezawa, Toshiaki, Ozaki, Katsuyuki, Nitani, Aya, Takabayashi, Chiyuki, Shimo-Oka, Tadashi
• Format: Journal Article
• Language: English
• Published: Los Angeles, CA SAGE Publications 01.01.2004; SAGE Publishing
• Subjects: Caco-2 Cells; Cell Culture Techniques; Cells, Cultured; Coculture Techniques; Collagen - chemistry; Connective Tissue; Epithelium; Fibroblasts - cytology; Gels; Humans; Mesoderm; Tissue Engineering - methods; Organoid; Scaffold; Vitrigel; Tissue engineering; Collagen; Three-dimensional culture
• ISSN: 0963-6897; 1555-3892
• DOI: 10.3727/000000004783983882

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 framework-embedded 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 anchorage-dependent 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 sheet-embedded 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.