Primary culture of mammalian taste epithelium

Abstract

Establishment of primary and immortalized cultures of many cell types has facilitated efforts to understand the signals involved in proliferation and differentiation and yielded tools to rapidly assay new molecules targeting specific receptor pathways. Taste cells are specialized sensory epithelial cells which reside within taste buds on the lingual epithelium. Only recently have successful culturing protocols been developed which maintain essential molecular and functional characteristics. These protocols provide a tractable tool to examine the molecular, regenerative, and functional properties of these unique sensory cells within a controlled environment. The method involves an enzymatic isolation procedure and standardized culture conditions, and may be applied to either dissected rodent tissue or human fungiform papillae obtained by biopsy. Human fungiform cells can be maintained in culture for more than seven passages, without loss of viability and with retention of the molecular and biochemical properties of acutely isolated taste cells. Cultured primary human fungiform papillae cells also exhibit functional responses to taste stimuli indicating the presence of taste receptors and at least some relevant signaling pathways. While the loss of the three-dimensional structure of the intact taste bud must be taken into consideration in interpreting results obtained with these cells, this culture protocol provides a useful model for molecular studies of the proliferation, differentiation, and physiological function of mammalian taste receptor cells.

Fig. 1

Attachment and morphology of cultured human fungiform taste cells. (a) Primary cell cultures grown on collagen type-1-coated plates were imaged after 2 days. (b) Cells from human fungiform papillae grew for up to 2–4 weeks under attached cell clusters. (c and d) Represent day 2 and 4 weeks after harvesting, respectively. During this period we did not observe growth of cells with the appearance of non-taste epithelial cells.

Fig. 2

Immunostaining of cultured human fungiform papillae cells showed the presence of taste cell-speci fic biomarkers. Images were acquired with a Leica TCS-SP2 confocal laser scanning microscope. (a and c) Transmission images of corresponding fields are shown on the left. (b) Immunoreactivity was observed for gustducin in about 60% of cultured cells. (d) Immunoreactivity for PLC-β₂ was also observed in about 25% of cultured cells (h). Nuclei of cells were stained as blue with DAPI. For controls, immunostaining with antibody-specific immunoglobulin demonstrated the absence of nonspecific immunoreactivity (data not shown). Scale bars = 50 μm.

Fig. 3

The expression taste cell marker mRNAs were demonstrated by RT-PCR. The expression taste cell markers of β-actin, gustducin, PLC β₂, T1R3, T2R5, and TRPM5 mRNA were shown in cultured human fungiform taste cells. The cDNA transcribed from total RNA was amplified with intron spanning-specific primers (Table 2). PCR products were found at expected size and confirmed by sequencing. Specific mRNA was not detected in control experiments without reverse transcriptase indicating no genomic DNA contamination. M = marker (100-bp division).

Fig. 4

Cultured taste cells responded to sweet, umami, and bitter stimuli. Changes in intracellular calcium levels ([Ca²⁺]_i) in cultured human fungiform taste cell were measured in 96-well plates using fura-2, a medium-throughput system. Stimuli were dissolved in modified Ringer solution and adjusted for pH and osmolarity. Graphs illustrate representative changes in [Ca²⁺]_i levels in individual cells during exposure to (a) denatonium benzoate (2 mM), (b) sucralose (1 mM), (c) Acesulfame-K (AceK, 250 ppm), (d) mono potassium glutamate (MPG, 3 mM).