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...

No abstract

A heparin-binding peptide was isolated from a proteolytic hydrolysate of bovine lactoferrin by affinity chromatography using an immobilized heparin column. Analysis of amino acid sequences at the N-terminus showed that this heparin-binding peptide is derived from the region beginning at the 17th amino acid residue of the bovine lactoferrin sequence. The molecular mass of this peptide was 3195.5 as measured by matrix-assisted laser desorption-time of flight mass spectrometry. This peptide is the same as the bactericidal peptide lactoferricin B. In an aqueous environment, this peptide displays mainly a beta-sheet structure and an unordered structure as assessed by measurements of circular dichroism spectra. When this peptide was mixed with heparin, a distinct spectral change was induced because of conformational alteration of the peptide. This spectral change was reversible. Analysis of data from peptide synthesis indicated that binding by the sequence Arg28-Met29-Lys30-Lys31 of bovine lactoferrin is significant and that there is a synergistic contribution from Lys18-Cys19-Arg20-Arg21, and Arg38-Arg39.

Soft and turbid collagen gel disks were previously converted into strong and transparent gel membranes utilizing a concept for the vitrification of heat-denatured of proteins. This novel stable and transparent gel has been termed ‘vitrigel’. By encompassing the collagen vitrigel membrane in a nylon frame, it can be easily handled with tweezers, and functions as an excellent scaffold for three-dimensional cell culture models, as cells can be cultured on both sides. Here, we investigated the molecular permeability of the collagen vitrigel membrane in a time course-dependent manner using glucose and serum proteins. Glucose penetrated through the collagen vitrigel membrane to the opposite side, and concentrations on each side were found to be equilibrated within 24 h. Serum proteins up to a molecular weight >100 kDa also gradually passed through the collagen vitrigel membrane. In addition, human microvascular endothelial cells (HMVECs) were cultured on one surface of the collagen vitrigel membrane with a nylon frame, and human dermal fibroblasts (HDFs) or HT-29 (a human colon carcinoma cell line) cells were cocultured on the opposite surface. Histomorphological observations revealed the formation of three-dimensional crosstalk models composed of HMVECs and HDFs or HMVECs and HT-29 cells. Resulting data suggest that the protein-permeable scaffold composed of the collagen vitrigel membrane is useful for the reconstruction and/or modeling of ‘crosstalk’ between two different cells types. Hereafter, such crosstalk models in vitro could be applied to research not only of paracrine factors, but also to epithelial- or endothelial-mesenchymal transitions.