Collagen vitrigel: a novel scaffold that can facilitate a three-dimensional culture for reconstructing organoids.
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 degrees 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.
Laboratory of Animal Cell Biology, National Institute of Agrobiological Sciences, Ikenodai 2, Tsukuba, Ibaraki 305-0901, Japan. t.takezawa@affrc.go.jp
Toshiaki
T
Takezawa
Katsuyuki
K
Ozaki
Aya
A
Nitani
Chiyuki
C
Takabayashi
Tadashi
T
Shimo-Oka
D018938:Caco-2 Cells; D018929:Cell Culture Techniques; D002478:Cells, Cultured; D018920:Coculture Techniques; D003094:Collagen / Q000737:chemistry*; D003238:Connective Tissue; D004848:Epithelium; D005347:Fibroblasts / Q000166:cytology; D005782:Gels; D006801:Humans; D008648:Mesoderm; D023822:Tissue Engineering / Q000379:methods*
D005782:Gels; D003094:Collagen