Abstract
Sweet, umami, bitter, salt, and sour are the five taste modalities; however, there is increasing evidence of a sixth taste modality related to the oro-sensory perception of dietary fatty acids. Fat taste is principally detected by cluster of differentiation 36 (CD36), G-protein-coupled receptor 120 (GPR120), and GPR40. Despite the high level of interest, it is very difficult to obtain ethical approval to isolate human taste bud cells (TBCs). Therefore, mouse TBCs are much sought after for in vitro studies. This study aimed to develop a method for the purification of CD36-expressing TBCs from mouse fungiform and circumvallate papillae.
After cervical dislocation, the tongue was removed, and an elastase/dispase enzyme mixture was injected under the epithelium and around the circumvallate papillae. The epithelium-containing taste buds were picked off and subjected to enzymatic digestion with the elastase and dispase mixture. The cells were isolated by using an anti-CD36 antibody coupled to phycoerythrin (PE) and anti-PE-antibodies coupled to magnetic beads. The mixture was then passed through a magnetic column in which the CD36-positive cells were retained.
The isolated cells were cultured for up to 5 days, and western blotting and quantitative reverse-transcription polymerase chain reaction (RT-qPCR) techniques revealed that purified cells expressed the receptors for CD36 and GPR120 as well as α-gustducin and phospholipase C (PLC) involved in downstream signal transduction. Using Fura-2-acetoxymethyl ester (Fura-2/AM), the selected positive cells were found to respond to dietary fatty acids via a CD36-induced increase in free intracellular Ca2+ concentrations. In conclusion, purified CD36-positive taste bud cells can be of great help for in vitro investigation of taste bud physiology and for studying the mechanisms of fat taste perception.
