We thank Aimee Myers and Esi Quayson for technical skills and help. This work was supported in part by NSF 0216310 and Givaudan Inc grant (NER).

1. Obtaining Human Fungiform Taste Papillae
<ol>
 <li>Remove four to eight human fungiform taste papillae from the dorsal surface of the anterior portion of the tongue using curved spring microscissors.</li>
 <li>Place immediately into an isolation solution (26 mM NaHCO3, 2.5 mM NaH2PO4, 20 mM glucose, 65 mM NaCl, 20 mM KCl, and 1 mM EDTA dissolved in nuclease-free water and filter sterilized).</li>
</ol>
2. Human Fungiform Taste Papillae Digestion
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<ol>
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No conflicts of interest declared.

We have maintained cells obtained from human fungiform taste papillae for over 8 passages, spanning a period of 12 months. These cultures generate new cells, many of which mature in vitro to the stage where they express several markers of mature taste bud cell types, in addition to key molecular and physiological properties of taste receptor cells. The presence of gustducin- and PLC-&beta;2 immunoreactivity in subsets of cultured cells is consistent with this conclusion. Most importantly, some cells responded to...

<table border="1">
<tbody>
 <tr>
 <td>Name of the reagent </td>
 <td>Company </td>
 </tr>
 <tr>
 <td>MCDB 123 </td>
 <td>Sigma-Aldrich </td>
 </tr>
 <tr>
 <td>Iscove's DMEM Medium </td>
 <td>MediaTech </td>
 </tr>
 <tr>
 <td>Elastase </td>
 <td>Sigma-Aldrich </td>
 </tr>
 <tr>
 <td>Pronase E </td>
 <td>Sigma-Aldrich </td>
 </tr>
 <tr>
 <td>Elastase </td>
 <td>Worthington </td>
 </tr>
 <tr>
 <td>Soy bean trypsin inhibitor </td>
 <td>Worthington </td>
 </tr>
 <tr>
 <td>Collagenase type 1 </td>
 <td>Worthington </td>
 </tr>
 <tr>
 <td>Rat tail collagen type 1 </td>
 <td>BD Sciences </td>
 </tr>
 <tr>
 <td>Fura-2 AM </td>
 <td>Molecular Probes </td>
 </tr>
 <tr>
 <td>Superscript First Strand Synthesis System for RT-PCR </td>
 <td>Invitrogen </td>
 </tr>
 <tr>
 <td>Leica TCS SP2 Spectral Confocal Microscope </td>
 <td>Leica Microsystems Inc. </td>
 </tr>
 <tr>
 <td>Discovery-1 imaging station and Metamorph software </td>
 <td>Molecular Devices </td>
 </tr>
 <tr>
 <td>Small fine-tip forceps and extra fine spring scissors </td>
 <td>Fine Science Tools </td>
 </tr>
 </tbody>
</table>

isolation and culture of human fungiform taste papillae cells

Taste cells are highly specialized, with unique histological, molecular and physiological characteristics that permit detection of a wide range of simple stimuli and complex chemical molecules contained in foods. In human, individual fungiform papillae contain from zero to as many as 20 taste buds. There is no established protocol for culturing human taste cells, although the ability to maintain taste papillae cells in culture for multiple cell cycles would be of considerable utility for characterizing the molecular, regenerative, and functional properties of these unique sensory cells. Earlier studies of taste cells have been done using freshly isolated cells in primary culture, explant cultures from rodents, or semi-intact taste buds in tissue slices1,2,3,4. Although each of these preparations has advantages, the development of long-term cultures would have provided significant benefits, particularly for studies of taste cell proliferation and differentiation. Several groups, including ours, have been interested in the development and establishment of taste cell culture models. Most attempts to culture taste cells have reported limited viability, with cells typically not lasting beyond 3-5 d5,6,7,8. We recently reported on a successful method for the extended culture of rodent taste cells9. We here report for the first time the establishment of an in vitro culture system for isolated human fungiform taste papillae cells. Cells from human fungiform papillae obtained by biopsy were successfully maintained in culture for more than eight passages (12 months) without loss of viability. Cells displayed many molecular and physiological features characteristic of mature taste cells. Gustducin and phospholipase C &beta;2, (PLC-&beta;2) mRNA were detected in many cells by reverse transcriptase-polymerase chain reaction and confirmed by sequencing. Immunocytochemistry analysis demonstrated the presence of gustducin and PLC-&beta;2 expression in cultured taste cells. Cultured human fungiform cells also exhibited increases in intracellular calcium in response to appropriate concentrations of several taste stimuli indicating that taste receptors and at least some of the signalling pathways were present. These results sufficient indicate that taste cells from adult humans can be generated and maintained for at least eight passages. Many of the cells retain physiological and biochemical characteristics of acutely isolated cells from the adult taste epithelium to support their use as a model taste system. This system will enable further studies of the processes involved in proliferation, differentiation and function of mammalian taste receptor cells in an in vitro preparation.
Human fungiform taste papillae used for establishing human fungiform cell culture were donated for research following proper informed consent under research protocols that were reviewed and approved by the IRB committee. The protocol (#0934) was approved by Schulman Associates Institutional Review Board Inc., Cincinnati, OH. Written protocol below is based on published parameters reported by Ozdener et al. 201110.

Watch this Scientific Journal Video about Isolation and Culture of Human Fungiform Taste Papillae Cells at JoVE.com

Isolation and Culture of Human Fungiform Taste Papillae Cells

We aimed to develop a reproducible protocol for isolating and maintaining long-term cultures of human fungiform taste papillae cells. Cells from human fungiform papillae obtained by biopsy were successfully maintained in culture for more than eight passages (12 months) without loss of viability.

Taste cells are highly specialized, with unique histological, molecular and physiological characteristics that permit detection of a wide range of simple ...

isolation-and-culture-of-human-fungiform-taste-papillae-cells

Research

JoVE Journal

Neuroscience

14.2K visualizações.  Monell Chemical Senses Center. Nosso objetivo foi desenvolver um protocolo reprodutível para isolar e manter a longo prazo culturas de células humanas fungiformes papilas gustativas. As células de humano papilas fungiformes obtidos por biópsia com êxito foram mantidas em cultura durante mais de oito passagens (12 meses) sem perda de viabilidade.