Establishing a primary taste-cell culture system as a basic analysis for new taste engineering

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We have established an in vitro culture system of taste bud cells from rat circumvallate papillae. The taste bud cells were adhesive and viable for over 3 days when cultured onto Matrigel-coated dishes in medium based on keratinocyte growth medium. The cells retained molecular markers for both the cytoskeleton and intracellular signalling such as cytokeratin 8 and phospholipase Cβ2. In addition, three intracellular signaling molecules, gustducin, phospholipase Cβ2, and inositol 1, 4, 5-trisphosphate receptor type 3, are expressed in the same correlation as those in vivo. We show that an exogenously expressed myc-tagged α1 A-adrenoceptor sorts into the plasma membrane, and transduces a ligand-dependent signal resulting in intracellular [Ca^<2+>] increase in about half of the infected cells.Under high Ca^<2+> condition (about 0.5 mM), the cells were tightly associated with each other and formed packed aggregates. Under low Ca^<2+> condition (below 1.1 mM), the cells were dispersed and had an elongated shape. These two forms were reversible and specifically dependent on Ca^<2+>. The results indicate that extracellular Ca^<2+> regulates cell shape and cell-to-cell adhesion of taste bud cells. We analyzed the subunits of laminin, a major component of the basement membrane, in circumvallate papillae of rat tongue by the method of RT-PCR and immunohistochemistry. As a result, the papillae contain β2 and γ1 as major β and γ subunits, respectively and three integrin β subunit species, β1, β4 and β5.