Hagfish exudate is a natural biological macromolecule made of keratin intermediate filament protein skeins and mucin vesicles. Here, we successfully examined this remarkable biomaterial as a substrate for three-dimensional (3D) cell culturing purposes. After the sterilization with chloroform vapor, Dulbecco's modified eagle medium was mixed with the exudate to rupture the vesicles and skeins; a highly soft, adherent, fibrous and biocompatible hydrogel was formed. A variety of cells, including Hela-FUCCI, NMuMG-FUCCI, 10T1/2 and C2C12, was cultured on the hagfish exudate. A remarkable 3D growth by ~2.5 folds after day 3, ~5 folds after day 5, ~10 folds after day 7 and ~15 folds after day 14 were seen compared to day one of culturing in the hagfish exudate scaffold. In addition, the phase contrast, fluorescent and confocal microscopy observations confirmed the organoid shape formation within the three-week culture. The viability of cells was almost 100% indicating the great in vitro and in vivo potential of this exceptional biomaterial with no cytotoxic effect.
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