This work was supported by NIH R21 DC03969-01 (to JGB). We thank Dr. Fritz Lischka for capturing single taste bud cells, Dr. Tauf Huque for performing the RT-PCR shown in Figure 4B and Mr. D. Bayley and Ms. S. Alarcon for technical assistance. M. Yanina Pepino is a fellow supported by a NIDAT32 DA07313 Washington University School of Medicine, St. Louis, MO (Cottler, L.B. PI).

1. Introduction and General Considerations
The general procedure involves local anesthesia of a small (1 cm2) area of the dorsal surface of the anterior tongue, followed by removal of a number (6-8) of fungiform (taste) papillae from that anesthetized area using a curved spring micro-scissors (McPherson-Vannas type #SR 5603, Roboz, Rockville, MD). The removed fungiform papillae are approximately 0.25 mm3 each. There is very little non-papillae (taste) epithelial tissue removed along with each papilla. New papillae form in this area after 3 to 5 weeks and regain functioning taste buds (Spielman and Brand, unpublished observations). There is no noticeable effect on taste or flavor of food after removal of this small amount of taste material. From studies that have counted individual taste buds in the adult human tongue, it can be estimated that the human tongue contains approximately 5000 taste buds. Our procedure removes at most 20 to 25 of these taste buds (on average, ~ 3.5 to 4 taste buds per fungiform papilla in human)2, or about 0.4% of the total. In addition there is no pain associated with the surgical loss of this tissue. One feels, of course, slight pain at the injection site only, as the needle enters the tongue. After anesthesia has worn off, there is occasionally mild discomfort for less than a day, but this discomfort is much less than what one experiences after accidentally "biting" ones tongue.
Over the past several years we have developed an alternative procedure that does not include anesthesia. The technique for tissue removal is identical for both. The only difference in the procedure is that the surgeon is no longer confined to a 1 cm2 diameter area where the anesthesia was addressed. Rather s/he can choose any site on the dorsal surface of the tongue where prominent papillae appear. The total number of removed papillae are limited to 6-8 in both techniques, with or without anesthesia If performed properly, the discomfort of removing 6-8 fungiform taste papillae is considered less of an issue when compared with the pain associated with the administering of local anesthesia A general survey of individuals who underwent both procedures, i.e., with and without anesthesia, showed that an overwhelming majority of subjects would dispense without local anesthesia in all future biopsies. The surgeon who developed this technique (AIS) and several colleagues taught by the author who performed biopsies without anesthesia, have tried it on themselves and testify that the discomfort is temporary and is considered less than that of biting one's tongue. Finally, dozens of individuals who agreed to be biopsied were asked if they would be willing to try removal of one papilla without anesthesia and if they judge it to be too painful they would undergo anesthesia, have all agreed to continue with the removal of papillae without anesthesia.
The entire biopsy should not take longer than 5-10 minutes. If anesthesia is employed another 2-3 minutes is added. Biopsy is typically carried out in the morning hours. Before biopsy, volunteers are instructed to eat a light meal two hours before the scheduled start time. At biopsy, written informed consent is obtained. Exclusion criteria at the time of biopsy include those with serious chronic illness, whether controlled by medication or not (e.g., any gastrointestinal diseases, neurodegenerative diseases, cancer, blood dyscrasias, allergy to the local anesthetics, etc.) those with heart rate greater than 100 or less than 60; systolic blood pressure greater than 145 or less than 100; diastolic blood pressure greater than 90 or less than 60, and the opinion of the surgeon that the subject can participate in the study.
Finally, it is important to consider that the biopsy procedure should be carried out in the same facility where tissue will be processed for examination since tissue integrity is rapidly lost as we have often noted by the declining quality of mRNA. To minimize these problems the tongue tissue should be processed as quickly as possible.
2. Subject Preparation
<ol>
 <li>The subject is seated in a dental chair or a seat with a headrest.</li>
 <li>Check heart rate and blood pressure at the time of biopsy. Should the subject fall outside of the range of blood pressure, heart rate or oral competence, the surgeon recommends to the subject that he/she make immediate appointment to see a physician.</li>
 <li>Inspect the oral cavity for general tissue health, with attention to indicators of xerostomia, geographic tongue or any tongue lesions. Any subject showing signs of oral disease, including tongue lesions or xerostomia are excluded from the study. Instruct the subject to rinse the mouth with water to remove all debris from the oral cavity. If necessary the tongue is cleaned with a disposable tongue scrapper to remove plaque or debris, which could be carried into the tongue during injection of the anesthesia. The surgeon uses sterile gauze pads in a gloved hand to hold the anterior ¼ of the tongue as it extends from the oral cavity.</li>
 <li>If anesthesia is used, a 1cc Tuberculin syringe with a 28 gauge x ½' needle containing 0.25 mL lidocaine (2%) with epinephrine (1:100,000) is slowly injected into one site just below the dorsal surface of the anterior portion of the tongue. The site of anesthesia is distal to the collection site to avoid interference by the lidocaine with subsequent studies. For comparison, the amount of lidocaine injected (0.25 mL) is about one quarter of the amount normally given for nerve block in dental procedures (0.8 mL to 1 mL). After insertion of the needle but before injection of the anesthetic, aspiration ensures that no blood vessel is struck. This precaution is taken even though it is anatomically unlikely that a large vessel will be struck close to the dorsal surface. The only arteries on the dorsal surface, the dorsal lingual arteries are small blood vessels primarily located more posterior to the anterior third, where the tissue harvesting occurs. The larger lingual arteries and the lingual veins run on the ventral surface of the tongue, far removed from the dorsal area receiving anesthetic 3. The 1 cm anesthetized area appears to be slightly pale compared with the surrounding tissue. This is due to the swelling of the tissue as it compresses the small blood vessels. It is also due to the vasoconstrictive effect of the epinephrine. After about one minute, a probe is used to test the area for numbness. If the area is not numb, a second injection (0.1 to 0.2 mL) may be given.</li>
</ol>
3. Tongue Papillae Removal Technique
<ol>
 <li>The anterior quarter of the tongue of the subject is wrapped into sterile gauze (Figure 1) and held in the left hand of the surgeon (if performing the biopsy with the right hand). One corner of the gauze is left open to allow the surgeon to wipe the tongue free of saliva. The tongue must be held firmly, but, of course, not so as to cause unnecessary discomfort. The tip of the index finger of the left hand is positioned under the tongue directly under the site where the biopsy is performed. By pushing the index finger upward, the tongue becomes stretched over the tip of the finger making the papillae stick out for easy biopsy. </li>
 <li>Criteria for choosing papillae for biopsy: Taste papillae are easily distinguishable from the surrounding filliform papillae by their shape and size. Fungiform papillae are round and somewhat isolated from the surrounding. In their immediate vicinity there is a circular space devoid of filliform papillae (Figure 2A, blue arrows). This space is about 1 mm in diameter. Another distinguishing feature of the fungiform papillae is an extended capillary arborization visible on top of most fungiform papillae of young subjects. Although arborization makes locating fungiform papillae easier, they are not necessarily good predictors of the existence of taste buds in that papilla. As a rule one starts harvesting toward the front end and moves further away from the tip of the tongue to avoid any capillary bleeding from obscuring the site of the next tissue collection.</li>
 <li>Once a good papilla has been identified, a spring micro-scissors (McPherson-Vannas, curved, sharp cutting edge 5 mm, comb, tip width 0.2 mm overall length 3" (RS-5603, Roboz) is used to clip it off. To effectively use the micro surgical scissors one should not use the tip of the blade for cutting; rather, as far inside the blade as possible (Figure 1, enlarged image and Figure 3), close to the point of intersection with the opposite blade. Removal of the papilla is achieved with one cutting move of the scissors (Figure 1). The further inside the open blades the targeted papilla appears, the more effective is the cutting. Care should be taken that the overall position of the scissors must be parallel with the dorsal surface of the tongue. </li>
 <li>Sometimes the tongue becomes dry as the surgeon is trying to identify a papilla for removal. Once dry, the ability of the operator to clearly see which is a fungiform papilla becomes more challenging and makes sliding of the blades over the papilla very difficult. An overly dry tongue usually makes the metal blade stick to the tongue making the delicate surgical move less controllable. Letting the subject close his/her mouth and have saliva come in contact with the surface of the tongue helps the process. Overly wet tongue is not good either. Using the corner of the gauze that one uses to hold the tongue helps in this process. </li>
 <li>Any capillary bleeding that does occur is treated by blotting with sterile gauze. Bleeding usually stops within a few minutes and does not interfere with additional tissue harvesting. Because the papillae extend above the epithelial surface of the tongue, and only the upper 3/4 of a fungiform papilla is removed (Figure 2B), that part of the fungiform showing taste buds, little bleeding is expected. </li>
 <li>Using a small Dumont forceps (Pattern type), the removed papillae are placed in chilled basic Ringer buffer ( 130 mM NaCl, 5 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 1 mM Na-Pyruvate, 20 mM HEPES-Na, pH 7.2; osmolality of ~ 305 milli osmols) for subsequent manipulations . It is imperative to NEVER pinch the papilla between the blades of a forceps. The tissue is very fragile and should be handled with extreme care.</li>
 <li>After removal of the papillae, the subject remains in the chair for ten to fifteen minutes. Each subject is told not to bite on their anesthetized tongue, but to keep their tongue at rest. Subjects should be visually monitored by the attending surgeon. Anesthesia is expected to wear off in approximately 30-60 minutes. Because the site of papillae removal may be slightly sore after the biopsy, they are instructed to be aware of spicy or hot (temperature) food, carbonated drinks or drinks that are either extremely cold or hot. A further indication is given to subjects that any slight reddish coloration of saliva is normal and will cease upon dissolution of the clot.</li>
 <li>Subjects are seen 30-40 days following removal of the papillae for evaluation of the site where the papillae were removed. By this time taste papillae should have completely regenerated.</li>
</ol>
5. Representative Results
Tissue collected can be used for immunohistochemistry, RT-PCR, in situ hybridization, calcium imaging, patch clamping and tissue culture. When performed correctly the appearance of the papillae are healthy looking, white, about 0.5-1 mm in diameter (Figure 2B).
Papillae collected for immunohistochemistry are processed based on existing and published protocols 4. A typical taste bud from a paraformaldehide-fixed taste papilla is shown in Figure 4A.
Biopsied papillae can also be enzymatically treated5 to achieve dissociation of taste bud cells. After the enzymatic dissociation, cells can be maintained for up to 4 hours in a humidified Petri dish at 4C. In this state they can be used for single cell PCR (Figure 4B), Ca-imaging, etc.
Cells show a variety of shapes, many being slender and bipolar (Figure 5A and 5B) as one would expect of a taste bud cell. When collecting cells or performing Ca-imaging, after about 20 minutes under a microscope, the cells begin to develop apoptotic blebs (Figure 6A and 6B, arrows) and many of them become round. Using a technique developed in our lab, cells were picked up individually using a patch-pipette, as shown in Figure 5A and 5B.
As mentioned above, papillae begin to regrow after 5 to 8 weeks. Figure 7A demonstrates the appearance of a tongue where 8 fungiform papillae were removed from left side a volunteer's tongue using no anesthesia. Notice 8 slight reddish spots, where the papillae were snipped off. No bleeding is observed after surgery. The present image was taken 10 minutes after the biopsy. Figure 7B is an image of the tongue of the same subject seen 40 days after the biopsy. Notice all papillae have regrown. To determine if the regenerated papillae are functional we asked our volunteer to agree to a second biopsy involving the exact same papillae. To correctly identify the appropriate papillae we were planning to re-harvest, we generated a grid (Figure 7C) that fits the 8 papillae that were removed in the first place and superimposed it over the tongue of the same volunteer after 40 days post initial biopsy. Aided by the contour of the tongue and the distance from the margin to the grid, once identified, we have removed a few of the same exact papillae and processed them for immunohistochemistry. Figure 4A is one of these papillae. Notice the outline of a taste bud (in yellow) and the immunopositive staining for Phospholipase Cβ2 (PLCβ2), a type 2 taste cell transduction-associated enzyme. These data demonstrate that using the biopsy procedure as described herein, the fungiform papillae regenerate sufficiently by 40 days to have also in place likely functional taste buds.
<img src="/files/ftp_upload/2201/2201fig1.jpg" alt="Tongue biopsy procedure; close-up image of sample extraction using forceps for histological analysis." data-altupdatedat="1747911167000" data-alt="Figure 1"> 
 Figure 1. The anterior quarter of the tongue of the subject is wrapped into sterile gauze and held in the left hand of the surgeon. Tongue must be held firmly. The enlarged image on the right shows the proper position of the scissors and the position of the papilla in relation to the blade.
<img src="/files/ftp_upload/2201/2201fig2.jpg" alt="Tissue biopsy process; surgical extraction tools; sample on needle; experiment method diagram." data-altupdatedat="1747911167000" data-alt="Figure 2"> 
 Figure 2. (A) Fungiform papillae are shown with blue arrows. These are round structures somewhat isolated from its surrounding and in their immediate vicinity there is a circular space devoid of filliform papillae. (B) Fungiform taste papilla on the tip of the spring micro-scissors seconds after it has been removed.
<img src="/files/ftp_upload/2201/2201fig3.jpg" alt="Biopsy technique diagram showing tissue removal with tweezers for medical examination." data-altupdatedat="1747911167000" data-alt="Figure 3"> 
 Figure 3. Self biopsy of a fungiform papilla using a mirror and no anesthesia. The inset illustrates the overall position of the scissors, which must be parallel with the dorsal surface of the tongue. If done properly and atraumatically, anesthesia is forgone by virtually all volunteers.
<img src="/files/ftp_upload/2201/2201fig4a.jpg" alt="Cellular structure analysis with highlighted regions; fluorescence microscopy image; research data." data-altupdatedat="1747911167000" data-alt="Figure 4a"> 
 Figure 4 A. Immunohistochemistry of a taste bud. A fungiform papilla visualized by Nomarski optics using antibodies to Phospholipase β2 and tagged with Red Fluorescent Protein. The outline of the taste bud and a few labeled taste cells are shown in yellow. <img src="/files/ftp_upload/2201/2201fig4b.jpg" alt="Gel electrophoresis result; DNA bands; molecular weight markers; lane analysis; 1.6 kB band." data-altupdatedat="1747911167000" data-alt="Figure 4b"> B. RT-PCR of a candidate sour taste receptor, Acid Sensing Ion Channel 1 (ASIC1). M= marker. Lanes 1 through 4 were single cell RT-PCR for ASIC1 from four different volunteers. After collection, fungiform papillae were enzymatically dissociated, cells individually picked up and expression of ASIC1 were done. Lanes 2 and 4 had normal expression of ASIC1.
<img src="/files/ftp_upload/2201/2201fig5.jpg" alt="Cellular microinjection microscope image showing needle insertion into cells for genetic material delivery." data-altupdatedat="1747911167000" data-alt="Figure 5"> 
 Figure 5. A. Image of single taste cells being picked up using a patch pipette. A large epithelial cell is visible in the lower left corner (5A). Two red blood cells are also visible above the patch pipette and behind the glass barrel. The taste cell is approximately 30-50 microns long and about 6 micron wide. For reference, a red blood cell is about 7-8 microns in diameter. B. shows another taste receptor cell surrounded by four epithelial cells. The taste cell looks crisp.
<img src="/files/ftp_upload/2201/2201fig6.jpg" alt="Microscopic examination; microscope image; cell morphology comparison; biological analysis." data-altupdatedat="1747911167000" data-alt="Figure 6"> 
 Figure 6. A. Images of cells that started to develop apoptotic blebs (ghost like round blebs attached to the taste cell, arrows). B. Cells are no longer harvested when their appearance changes as shown here.
<img src="/files/ftp_upload/2201/2201fig7.jpg" alt="Oral wound healing, comparison, 10 minutes vs. 40 days, clinical observation, tongue regeneration." data-altupdatedat="1747911167000" data-alt="Figure 7"> 
 Figure 7. A. Ten minutes after removal of 8 taste papillae using no anesthesia. The surgical sites are visible as slightly reddish spots. No bleeding is noticed. B. The same tongue 40 days after biopsy. Notice all papillae regenerated. To help identify the removed papillae as regrown, a grid was created with the site on the right side picture and shifted over the regenerated papillae on the left side image. C. The regenerated papillae appeared in the exact same spot. Harvesting papillae again from this group followed by immunohistochemistry demonstrated that the regenerated papillae contained functional taste cells (See Figure 4A).

<ol>
	<li>Breslin, P. A., Spector, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mammalian+taste+perception.">Mammalian taste perception.</a> Curr Biol. 26 (4), R148-R155 (2008).</li><li>Miller, I., Doty, R. I., Dekker, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anatomy+of+the+peripheral+taste+system.">Anatomy of the peripheral taste system.</a> Handbook of Olfaction and Gustation. , 521-547 (1995).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Figure+12.67.">Figure 12.67.</a> Gray's Anatomy: The Anatomical Basis of Medicine and Surgery. , (1995).</li><li>Huque, T., Cowart, B. J., Dankulich-Nagrudny, L., Pribitkin, E. A., Bayley, D. L., Spielman, A., Feldman, I., S, R., Mackler, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sour+ageusia+in+two+individuals+implicates+ion+channels+of+the+ASIC+and+PKD+families+in+human+sour+taste+perception+at+the+anterior+tongue.">Sour ageusia in two individuals implicates ion channels of the ASIC and PKD families in human sour taste perception at the anterior tongue.</a> PLoS One. 4 (10), e7347-e7347 (2009).</li><li>MacDonald, J. F., Mody, I., Salter, M. W., Spielman, A. I., Spielman, A. I., Brand, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patch-clamping+of+taste+cells+in+mouse.">Patch-clamping of taste cells in mouse.</a> Experimental Cell Biology of Taste and Olfaction. , 329-332 (1995).</li></ol>

No conflicts of interest declared.

One notable advantage to using human cells is their specificity. The procedure described here in detail provides tongue tissue for isolation of taste receptor cells from humans. These taste cells can be used for molecular studies, seeking to discover molecules important for transduction of taste information, and for calcium imaging studies that monitor taste cell activity to stimuli. Additionally, tongue tissue provided by this procedure can be analyzed with various techniques such as immunohistochemistry, in situ hybridization, and allows capturing individual cells for molecular procedures such as Polymerase Chain Reaction (PCR), including Real Time Quantitative Single Cell PCR. Because this procedure can be performed on individuals whose sense of taste can be psychophysically evaluated prior to biopsy, it is possible to get a clear picture of the molecular details responsible for taste abnormalities (for example, see Huque et al. 2009). This biopsy procedure allows production of a viable and metabolically intact single cell preparation enriched in cells from taste buds and thus the ability to perform patch clamp recordings on these cells. Using this technique, future studies could not only increase our understanding of taste transduction processes unique to man but also decipher molecular mechanisms underlying taste abnormalities in disease or medication-induced states and develop regimens to treat these abnormalities.
Finally, the results of our observations strongly indicate that the presence of "holes," often noted on fungiform papillae close to arborizations and generally considered to be taste bud pores, are, in fact, not good predictors of the existence of taste buds. Just what the nature of these holes is, or what functions they may perform, we do not know.
In 8 years since we started this procedure, we have repeatedly collected from several dozen volunteers over 4000 taste papillae. Two adverse incidence, both associated with the local anesthesia led to a temporary swelling of the tongue, but resolved within the next 48 hours without adverse effects. If done properly, under sterile and atraumatic conditions, this method is safe and effective.

<table><tbody><tr><td>McPherson-Vannas. Curved, sharp cutting edge 5mm. Comb, tip width 0.2 mm overall length 3&quot;</td><td>Roboz Surgical Instruments Co.</td><td>RS-5603, Roboz</td><td></td></tr><tr><td>Small Dumont forceps</td><td>Roboz Surgical Instruments Co.</td><td>RS-4915, Roboz</td><td></td></tr></tbody></table>

technique to collect fungiform (taste) papillae from human tongue

The sense of taste is critical for human life. It informs the body about the quality of food that will be potentially ingested and stimulates metabolic processes that prepare the alimentary canal for digestion. Steady progress is being made towards understanding the early biochemical and molecular events underlying taste transduction (for a review, Breslin and Spector, 20081). However, progress to date has largely resulted from animal models. Yet, since marked differences in receptor specificity and receptor density vary among species, human taste transduction will only be understood by using human taste tissue. Here we describe a biopsy technique to collect human fungiform papillae, visible as rounded pink anterior structures, about 0.5 mm in diameter that contain taste buds. These biopsied papillae are used for several purposes including the isolation of viable taste bud cells, in situ hybridization, immunohistochemistry and, through techniques of molecular biology, the identification of taste-specific novel proteins.

Watch this Scientific Journal Video about Technique to Collect Fungiform Taste Papillae from Human Tongue at JoVE.com

Technique to Collect Fungiform Taste Papillae from Human Tongue

Knowledge of molecular mechanisms underlying gustatory transduction has recently enjoyed significant advances, largely due to using animal models. However, the wide diversity in taste sensitivity and specificity among mammals warrants studies in human tissue. We describe a biopsy technique to collect living taste cells from the papillae on human tongue.

Technique to Collect Fungiform (Taste) Papillae from Human Tongue

The sense of taste is critical for human life. It informs the body about the quality of food that will be potentially ingested and stimulates metabolic ...

technique-to-collect-fungiform-taste-papillae-from-human-tongue

Nanyang Technological University

Research

JoVE Journal

Medicine

28.9K Views.  College of Dentistry, New York University. Kennis van de moleculaire mechanismen die ten grondslag liggen smaak transductie heeft onlangs genoten van belangrijke vooruitgang, vooral door het gebruik van dierlijke modellen. Echter, de grote verscheidenheid in smaak sensitiviteit en specificiteit bij zoogdieren garandeert studies in menselijk weefsel. Beschrijven we een biopsie techniek om levende cellen te verzamelen smaak van de papillen op de menselijke tong.

Video: Technique to Collect Fungiform Taste Papillae from Human Tongue

Fabrication of Tongue Extracellular Matrix and Reconstitution of Tongue Squamous Cell Carcinoma In Vitro

In order to construct an effective and realistic model for tongue squamous cell carcinoma (TSCC) in vitro, the methods were created to produce decellularized tongue extracellular matrix (TEM) which provides functional scaffolds for TSCC construction. TEM provides an in vitro niche for cell growth, differentiation, and cell migration. The microstructures of native extracellular matrix (ECM) and biochemical compositions retained in the decellularized matrix provide tissue-specific niches for anchoring cells. The fabrication of TEM can be realized by deoxyribonuclease (DNase) digestion accompanied with a serious of organic or inorganic pretreatment. This protocol is easy to operate and ensures high efficiency for the decellularization. The TEM showed favorable cytocompatibility for TSCC cells under static or stirred culture conditions, which enables the construction of the TSCC model. A self-made bioreactor was also used for the persistent stirred condition for cell culture. Reconstructed TSCC using TEM showed the characteristics and properties resembling clinical TSCC histopathology, suggesting the potential in TSCC research.

Fabrication of Tongue Extracellular Matrix and Reconstitution of Tongue Squamous Cell Carcinoma In Vitro

A method is shown here for the preparation of the tongue extracellular matrix (TEM) with efficient decellularization. The TEM can be used as functional scaffolds for the reconstruction of a tongue squamous cell carcinoma (TSCC) model under static or stirred culture conditions.

In order to construct an effective and realistic model for tongue squamous cell carcinoma (TSCC) in vitro, the methods were created to produce ...

Cancer Research

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.

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 ...

Neuroscience

Measuring Oral Fatty Acid Thresholds, Fat Perception, Fatty Food Liking, and Papillae Density in Humans

Emerging evidence from a number of laboratories indicates that humans have the ability to identify fatty acids in the oral cavity, presumably via fatty acid receptors housed on taste cells. Previous research has shown that an individual&#39;s oral sensitivity to fatty acid, specifically oleic acid (C18:1) is associated with body mass index (BMI), dietary fat consumption, and the ability to identify fat in foods. We have developed a reliable and reproducible method to assess oral chemoreception of fatty acids, using a milk and C18:1 emulsion, together with an ascending forced choice triangle procedure. In parallel, a food matrix has been developed to assess an individual&#39;s ability to perceive fat, in addition to a simple method to assess fatty food liking. As an added measure tongue photography is used to assess papillae density, with higher density often being associated with increased taste sensitivity.

Oral chemoreception of fatty acids and the association with diet and fatty food preferences may enable the identification of mechanisms involved with the development of obesity and why dietary changes may be difficult for many individuals.

Emerging evidence from a number of laboratories indicates that humans have the ability to identify fatty acids in the oral cavity, presumably via fatty ...

Denver Papillae Protocol for Objective Analysis of Fungiform Papillae

The goal of the Denver Papillae Protocol is to use a dichotomous key to define and prioritize the characteristics of fungiform papillae (FP) to ensure consistent scoring between scorers. This protocol builds off of a need that has arisen from the last two decades of taste research using FP as a proxy for taste pore density. FP density has historically been analyzed using Miller &#38; Reedy&#8217;s 1990 characterizations of their morphology: round, stained lighter, large, and elevated. In this work, the authors forewarned that stricter definitions of FP morphology needed to be outlined. Despite this call to action, follow up literature has been scarce, with most studies continuing to cite Miller &#38; Reedy&#8217;s original work. Consequently, FP density reports have been highly variable and, combined with small sample sizes, may contribute to the discrepant conclusions on the role of FP in taste sensitivity. The Genetics of Taste Lab explored this apparent inconsistency in counting and found that scorers were individually prioritizing the importance of these characteristics differently and had no guidance for when a papilla had some, but not all, of the reported qualities of FP. The result of this subjectivity is highly variable FP counts of the same tongue image. The Denver Papillae Protocol has been developed to remedy this consequence through use of a dichotomous key that further defines and prioritizes the importance of the characteristics put forth by Miller &#38; Reedy. The proposed method could help create a standard way to quantify FP for researchers in the field of taste and nutritional studies.

Here we present a protocol to measure fungiform papilla density from digital photographs. This method builds prioritization and objective characteristic metrics into the original descriptive work on fungiform papillae by Miller &#38; Reedy (1990).

The goal of the Denver Papillae Protocol is to use a dichotomous key to define and prioritize the characteristics of fungiform papillae (FP) to ensure ...

Taste Exam: A Brief and Validated Test

The emerging importance of taste in medicine and biomedical research, and new knowledge about its genetic underpinnings, has motivated us to supplement classic taste-testing methods in two ways. First, we explain how to do a brief assessment of the mouth, including the tongue, to ensure that taste papillae are present and to note evidence of relevant disease. Second, we draw on genetics to validate taste test data by comparing reports of perceived bitterness intensity and inborn receptor genotypes. Discordance between objective measures of genotype and subjective reports of taste experience can identify data collection errors, distracted subjects or those who have not understood or followed instructions. Our expectation is that fast and valid taste tests may persuade researchers and clinicians to assess taste regularly, making taste testing as common as testing for hearing and vision. Finally, because many tissues of the body express taste receptors, taste responses may provide a proxy for tissue sensitivity elsewhere in the body and, thereby, serve as a rapid, point-of-care test to guide diagnosis and a research tool to evaluate taste receptor protein function.

This protocol measures human taste responses and includes a brief anatomical assessment, a short taste test, and a validation method using the subject&#39;s reported sensation and taste receptor genotype.

The emerging importance of taste in medicine and biomedical research, and new knowledge about its genetic underpinnings, has motivated us to supplement ...

Whole-Mount Staining, Visualization, and Analysis of Fungiform, Circumvallate, and Palate Taste Buds

Taste buds are collections of taste-transducing cells specialized to detect subsets of chemical stimuli in the oral cavity. These transducing cells communicate with nerve fibers that carry this information to the brain. Because taste-transducing cells continuously die and are replaced throughout adulthood, the taste-bud environment is both complex and dynamic, requiring detailed analyses of its cell types, their locations, and any physical relationships between them. Detailed analyses have been limited by tongue-tissue heterogeneity and density that have significantly reduced antibody permeability. These obstacles require sectioning protocols that result in splitting taste buds across sections so that measurements are only approximated, and cell relationships are lost. To overcome these challenges, the methods described herein involve collecting, imaging, and analyzing whole taste buds and individual terminal arbors from three taste regions: fungiform papillae, circumvallate papillae, and the palate. Collecting whole taste buds reduces bias and technical variability and can be used to report absolute numbers for features including taste-bud volume, total taste-bud innervation, transducing-cell counts, and the morphology of individual terminal arbors. To demonstrate the advantages of this method, this paper provides comparisons of taste bud and innervation volumes between fungiform and circumvallate taste buds using a general taste-bud marker and a label for all taste fibers. A workflow for the use of sparse-cell genetic labeling of taste neurons (with labeled subsets of taste-transducing cells) is also provided. This workflow analyzes the structures of individual taste-nerve arbors, cell type numbers, and the physical relationships between cells using image analysis software. Together, these workflows provide a novel approach for tissue preparation and analysis of both whole taste buds and the complete morphology of their innervating arbors.

This paper describes methods for tissue preparation, staining, and analysis of whole fungiform, circumvallate, and palate taste buds that consistently yield whole and intact taste buds (including the nerve fibers that innervate them) and maintain the relationships between structures within taste buds and the surrounding papilla.

Taste buds are collections of taste-transducing cells specialized to detect subsets of chemical stimuli in the oral cavity. These transducing cells ...

&#181;Tongue: A Microfluidics-Based Functional Imaging Platform for the Tongue In Vivo

Intravital fluorescence microscopy is a tool used widely to study multicellular dynamics in a live animal. However, it has not been successfully used in the taste sensory organ. By integrating microfluidics into the intravital tongue imaging window, the &#181;Tongue provides reliable functional images of taste cells in vivo under controlled exposure to multiple tastants. In this paper, a detailed step-by-step procedure to utilize the &#181;Tongue system is presented. There are five subsections: preparing of tastant solutions, setting up of a microfluidic module, sample mounting, acquiring functional image data, and data analysis. Some tips and techniques to solve the practical issues that may arise when using the &#181;Tongue are also presented.

 &#181;Tongue: A Microfluidics-Based Functional Imaging Platform for the Tongue In Vivo

The article introduces the &#181;Tongue (microfluidics-on-a-tongue) device for functional taste cell imaging in vivo by integrating microfluidics into an intravital imaging window on the tongue.

Intravital fluorescence microscopy is a tool used widely to study multicellular dynamics in a live animal. However, it has not been successfully used in ...

Generation and Characterization of Murine Oral Mucosal Organoid Cultures

The mucous lining covering the inside of our mouth, the oral mucosa, is a highly compartmentalized tissue and can be subdivided into the buccal mucosa, gingiva, lips, palate, and tongue. Its uppermost layer, the oral epithelium, is maintained by adult stem cells throughout life. Proliferation and differentiation of adult epithelial stem cells have been intensively studied using in vivo mouse models as well as two-dimensional (2D) feeder-cell based in vitro models. Complementary to these methods is organoid technology, where adult stem cells are embedded into an extracellular matrix (ECM)-rich hydrogel and provided with a culture medium containing a defined cocktail of growth factors. Under these conditions, adult stem cells proliferate and spontaneously form three-dimensional (3D) cell clusters, the so-called organoids. Organoid cultures were initially established from murine small intestinal epithelial stem cells. However, the method has since been adapted for other epithelial stem cell types. Here, we describe a protocol for the generation and characterization of murine oral mucosal organoid cultures. Primary epithelial cells are isolated from murine tongue tissue, embedded into an ECM hydrogel, and cultured in a medium containing: epidermal growth factor (EGF), R-spondin, and fibroblast growth factor (FGF) 10. Within 7 to 14 days of initial seeding, the resulting organoids can be passaged for further expansion and cryopreservation. We additionally present strategies for the characterization of established organoid cultures via 3D whole-mount imaging and gene-expression analysis. This protocol may serve as a tool to investigate oral epithelial stem cell behavior ex vivo in a reductionist manner.

We present a method for the generation and characterization of oral mucosal organoid cultures derived from the tongue epithelium of adult mice.

The mucous lining covering the inside of our mouth, the oral mucosa, is a highly compartmentalized tissue and can be subdivided into the buccal mucosa, ...

Biology

The Tongue and Taste Buds

The surface of the tongue is covered with various small bumps called papillae, which either distribute what has been ingested (filiform papillae) or contain the sensory taste (or gustatory) receptor cells (fungiform, circumvallate, and foliate papillae). Embedded within each taste-related papilla are the taste buds&mdash;clusters of 30 to 100 gustatory receptor cells.

Gustatory receptor cells extend finger-like projections called gustatory hairs (or microvilli) into a region known as the taste pore. Here, many of the cells contain receptors that detect different tastants&mdash;the molecules that can be tasted. The average number of taste buds varies significantly among individuals, with estimates ranging from 2,000-10,000 taste buds. Taste cells have a lifespan of about 10-14 days and are continually replaced. Thus, each taste bud contains taste cells at different stages of development.

<h4>Different Types of Taste Papillae</h4>

Aside from the filiform papillae, which do not contain taste buds, the mushroom-shaped fungiform papillae are the most numerous. Fungiform papillae are predominantly located on the anterior two-thirds of the tongue and contain between one and eight taste buds each.

In contrast, the other two types of papillae&mdash;circumvallate and foliate&mdash;contain more than 100 taste buds per papilla. Circumvallate papillae, the largest type, are located at the back of the tongue in &ldquo;V&rdquo;-like formation. Nearby, on the sides of the tongue, are the foliate papillae.

<h4>The Myth of the Tongue&rsquo;s Taste Map</h4>

The work of many scientists, including Collings, Yanagisawa, and colleagues, indicates that the five basic tastes can be perceived anywhere across the tongue. Thus, distinct tastes are not restricted to particular regions, as typically indicated on tongue maps.

The surface of the tongue is covered with various small bumps called papillae, which either distribute what has been ingested (filiform papillae) or ...

Education

JoVE Core

Human Biology

Sensory Systems

KEY TERMS AND CONCEPTS

Isolation of Circumvallate Papillae (CVP) Epithelium from Mouse Model: An Enzymatic Procedure to Separate CVP Epithelium from Mouse Tongue

After euthanizing an adult mouse, use large sterile dissection scissors to cut the cheeks and break the jaw. Then, lift the tongue and cut the lingual frenulum to separate the tongue from the floor of the oral cavity. Cut the tongue out and collect it in sterile ice-cold dPBS with calcium and magnesium.
Under a dissecting microscope, remove and discard the anterior tongue by cutting just anterior of the intermolar eminence with a razor blade. Then, using a delicate task wipe to remove any hair and excess liquid from the posterior tongue.
Next, fill a 1-milliliter syringe with 200 to 300 microliters of injection enzyme solution and insert a 30-gauge, 1/2 inch needle just above the intermolar eminence until just interior of the circumvallate papillae or CVP. Inject the enzyme solution underneath and at the lateral edges of the CVP between the epithelium and the underlying tissues. Withdraw the syringe slowly and continuously from the tongue as the solution is being injected.
Incubate the tongue in sterile calcium/magnesium-free dPBS at room temperature for precisely 33 minutes. Using extra fine dissection scissors, make small cuts in the epithelium bilaterally and just anterior of the CVP. Then, gently peel the epithelium by lifting it with fine forceps. Once the trench epithelium is free of the underlying connective tissue, place it in a 2-milliliter microcentrifuge tube pre-coated with FBS.

Technique to Collect Fungiform (Taste) Papillae from Human Tongue

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