Figure 3. Lgr5+ cell-derived taste bud organoid culture. Source: 67. Reproduced with permission of Proc Natl Acad Sci USA. (a) Lgr5+ cells in the taste papilla. (b) Results of FACS sorting of Lgr5+ cells. (c) Single Lgr5+ cell (green). (d) Representative images of taste bud organoids at different days in culture.
3.2 Tissue-derived approach
The commonly used approach of cultivating taste bud organoids is to obtain the digestive juice of taste bud tissue and then cultivate it. Both mice and rats can be used as research objects to obtain taste buds for the cultivation of taste bud organoids. Injecting collagenase and dispase into the tongue to obtain the tongue epithelium, then separate the CV papillae, as well as part of the non-taste epithelium without taste buds[73]. [68]After further digesting the obtained taste papilla tissue with trypsin, it was mixed with matrigel and inoculated in a culture plate, and organoids were grown from these tissues (Figure 4). Then, the expression levels of taste bud lineage markers mRNA and proteins as well as immunofluorescence be used to characterize their physiological characteristics. The greatest convenience of the tissue-derived taste bud organoid culture approach is that there is no need to screen and culture specific cells and omit the step of single-cell sorting. In addition, the tissues contain other types of cells, which can better simulate the physiological conditions in the body and provide a living environment closer to the living body for the cultivation of organoids. Taste bud organoids from FUCCI2 mice in which mCherry-hCdt1 (red fluorescence) is expressed during G1 phase while mVenus-hGem (green fluorescence) is expressed during the S/G2/M phase of the cell cycle[68]. Another study showed that Trpv4 deficiency reduced sensitivity to sourness and the expression of type III cells in taste bud organoid[25]. These results can well prove that taste bud organoids derived from tissues can be used in research.