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Ozdener H.,Monell Chemical Senses Center | Spielman A.I.,New York University | Rawson N.E.,AFB International
Journal of Visualized Experiments | Year: 2012

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 slices 1,2,3,4. 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 d 5,6,7,8. We recently reported on a successful method for the extended culture of rodent taste cells 9. 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 β2, (PLC-β2) 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-β2 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. 2011 10. © JoVE 2006-2012. All Rights Reserved. Source


Rawson N.E.,AFB International | Ozdener M.H.,Monell Chemical Senses Center
Methods in Molecular Biology | Year: 2013

The central cell type involved in the initial perception of odors and transduction of the sensory signal are the olfactory receptor neurons (ORNs) located in the olfactory neuroepithelium of the nasal cavities. The olfactory epithelium is a unique system similar to the neuroepithelium of the embryonic neural tube, in which new neurons are continually generated throughout adult life. Olfactory neurons are derived from precursor cells that lie adjacent to the basal lamina of the olfactory epithelium; these precursor cells divide several times and their progeny differentiate into mature sensory neurons throughout life. Thus, the human olfactory epithelium has the potential to be used as a tool to examine certain human disorders resulting from abnormal development of the nervous system. This chapter presents methods for primary culture of human ORNs, which have been used successfully by multiple investigators. The protocol provides a consistent, heterogeneous cell population, which demonstrates functional responses to odorant mixtures and exhibits a complex neuronal phenotype, encompassing receptors and signaling pathways pertinent to both olfaction and other aspects of CNS function. These cultured neural cells exhibit neurotransmitter pathways important in a number of neuropsychiatric disorders, and the ability to culture cells from living human subjects provides a tool for assessing cellular neuropathology at the individual patient level. © 2013 Springer Science+Business Media, LLC. Source


Rawson N.,AFB International | Quraishi A.,American Gas Association | Bruno T.J.,U.S. National Institute of Standards and Technology
Journal of Research of the National Institute of Standards and Technology | Year: 2011

Since the days of the alchemist, the observation that some substances have a smell while other substances do not has been a source of fascination. The sense of smell, or olfaction, is our least understood sense, however it is important for many human functions, including digestion, food selection and hazard avoidance. The detailed explanation of why individual chemicals (called odorants) might have a particular smell is still elusive. The situation with mixtures of odorants is even more complex and interesting. A number of distinct odorant mixture phenomena have been documented. Odorant suppression (sometimes called masking), conjugation (as described first by Zwaadermaker) and cross-adaptation are among a collection of such phenomena. They are related to the differential effects that one odorant species will have when mixed with another. Masking is a term that describes situations in which one odorant can overpower the sensation of another. There may be profound technological implications in a number of industrial sectors, most prominently in the fuel gas sector. Here, masking is suspected when the odorant that is added to natural gas can be detected by analytical instrumentation, but cannot be properly detected by an observer with a normal sense of smell. Note that this phenomenon is distinct from odor fade, which more properly describes a decrease in the concentration of an odorant rather than a decrease, disappearance or qualitative change in the perception of the odor in the absence of a change in absolute concentration. Anecdotal descriptions of masking events in the natural gas industry have persisted for over a decade, with the frequency of such events on the rise. Pursuant to the philosophy that the technological problem cannot be addressed until the basic science is understood, NIST, in collaboration with the American Gas Association (AGA), sponsored a workshop that brought together olfactory scientists and natural gas operations personnel in an effort to achieve a common understanding and identify critical research questions. This document is a summary of that workshop, and most importantly, a compendium of the findings and recommendations that resulted from the meeting. Source


Patent
Afb International | Date: 2014-03-06

A family of novel feline bitter taste receptors, referred to as feline TAS2R (fTAS2R), are disclosed herein. Isolated polynucleotides encoding the novel feline bitter taste receptors and chimeric polypeptides are also disclosed, as are expression vectors and host cells for expression of the novel feline bitter taste receptors. Methods of identifying compounds that bind to the novel feline bitter taste receptors and modulate their activity are disclosed.


Ozdener M.H.,Monell Chemical Senses Center | Rawson N.E.,AFB International
Methods in Molecular Biology | Year: 2013

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. © 2013 Springer Science+Business Media, LLC. Source

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