Isolation and Culture of Human Fungiform Taste Papillae Cells

1. Obtaining Human Fungiform Taste Papillae

1. Remove four to eight human fungiform taste papillae from the dorsal surface of the anterior portion of the tongue using curved spring microscissors.
2. 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).

2. Human Fungiform Taste Papillae Digestion

Human fungiform taste cell papillae can be dissociated using two different enzymatic digestion protocols with slight differences.

1. Incubate fungiform papillae in isolation solution with pronase (1 mg/ml, Sigma Aldrich) and elastase (1 mg/ml Sigma Aldrich) at room temperature for 30-45 min (protocol 1).
2. Incubate fungiform papillae with Collagenase type I (550 U/mL, from Worthington) + elastase (10 U/mL, from Worthington) + Trypsin inhibitor (0.9 mg/mL, from Worthington) in 2 ml calcium free Ringer solution in 35 °C water bath with circulation for 30 minutes and gentle oxygenation with 95% O₂ / 5% CO₂. (Alternative method for digestion, protocol 2)
3. After incubation, wash papillae with ringer and triturate the papillae with fire polished glass Pasteur pipette for 10 times.
4. Centrifuge it for 3 minutes at 2500 rpm at RT and remove isolation solution and add 1 ml of taste cell culture medium.
5. Transfer digested fungiform papillae into glass dish.
6. Dissect fungiform papillae gently with surgical razor.
7. Add 250 μl of dissected papillae into cloning cylinder onto rat tail collagen type-1 coated coverslip.
8. Add 1 ml of taste cell culture medium into each well.

3. Culturing of Human Fungiform Taste Papillae Cells

1. Incubate plate at 36 °C in a humidified incubator containing 5% CO₂.
2. Place in an incubator undisturbed for 2 days prior to the first change of complete medium. Taste cells will eventually bind to the coated coverslip, although it may not be clearly visible in 1 to 3 days.
3. Remove cloning cylinder from plate and medium completely and add 1 ml of taste cell medium into each well.
4. Replace 1/3 of medium every 6-7 days. Do not change medium more often and/or completely.
5. Check cell growth under microscope every other day. Most of the new cells grow underneath cell clusters. Cell clusters usually detach after 2-3 weeks in culture leaving the newly generated cells.
6. Once 40-50% of the cloning cylinder is covered in expanding taste cells, trypsinize cells using 0.25% w/v trypsin/EDTA for 2-3 minutes at 36 °C.
7. Transfer cells from wells into 15 ml tubes, add 3 volumes of taste cell culture medium followed by centrifugation at 3000 rpm for 5 minutes at room temperature.
8. Remove supernatant and resuspend cells with 1 ml of taste cell medium.

4. Propagation of Human Fungiform Taste Papillae Cells

1. Transfer cells into T25 plate and add 4 ml of taste cell medium (passage 0). Maintain cells at 36 °C in a humidified incubator containing 5% CO₂.
2. Replace 1/3 of medium every 6-7 days until cultured taste cells have reached 100% confluence. At this time, to enable taste cells to harvest, wash cells once with sterile PBS then trypsinize cells using 0.25% w/v trypsin/EDTA for 2-3 minutes at 36 °C.
3. After centrifugation as described above, resuspend in complete taste cell medium and transfer cells to fresh T-75 flasks (passage 1).
4. Replace 1/3 of medium every 6-7 days. Do not change medium more often and/or completely.
5. Repeat step 4.3 and 4.4 when cells have reached close to 100% confluence. Split cells to no more than 1:4 dilution in a T75 flask for maintaining adequate growth of the cells over time.

5. Freezing and Thawing Cultured Human Fungiform Taste Papillae Cells

1. To freeze stocks of primary taste cells, after trypsinization (step 4.2), add 3 ml complete taste cell medium and transfer cells to sterile 15 ml conical centrifuge tubes. Centrifuge at 2500 rpm for 5 min at room temperature.
2. Carefully remove the supernatant and gently resuspend cells with appropriate volume of freezing medium containing 95% Fetal Bovine serum (FBS) and 5% DMSO.
3. Transfer cells to labeled, sterile cryovials, cap tightly, and place in a freezing container containing isopropanol. Place into a -80 °C freezer for at least one day prior to transferring indefinitely to liquid nitrogen.
4. To thaw a vial of frozen primary human fungiform taste papillae cells, place at 37 °C water bath until just thawed (approximately 1 minute), while working in a laminar-flow cell culture hood.
5. Transfer cells and freezing medium to a sterile 15 ml conical centrifuge tube and add 5 ml of taste cell culture medium.
6. Centrifuge cells at 2500 rpm for 5 min at room temperature. Carefully discard supernatant, gently resuspend cells with complete taste cell culture medium, and transfer to a sterile T-25 tissue culture flask.
7. Continue to culture cells according to Step 3 to 4.

6. Representative Results

Attempts to grow taste biopsies in culture were successful 90% of the time. A few days after biopsy and dissociation, cells typically began to grow and migrate outwards from the pieces of dissected fungiform tissue that remained after the dissociation procedure. Although individual cells were visible 24-48 hr after plating (Figure 1A), cells grew for up to 2-3 weeks under attached cell clusters, occasionally generating daughter cells (Figure 1B). From biopsying 4-8 human fungiform papillae, initial isolates of primary human fungiform taste papillae cells reach approximately 2000 – 8000 cells in 4 weeks in culture. Expansion in T25 cell culture flasks (passage 0) for additional 3-4 weeks will result in having human fungiform taste papillae cells in culture adapted (Figure 1C). Primary human fungiform taste papillae cells can be further expanded in culture to yield upwards of at least a million cells by passage-1 in 3-4 weeks (Figure 1D). Primary taste cells will continue to grow for more than 8 passages.

The morphology of the cultured human fungiform taste papillae cells in culture was similar to cultured chemosensory cells described previously^11,12.. Most cultured human fungiform taste papillae cells maintained their original compact appearance of cell bodies with or without one or more processes up to 15-30 days. After they reached confluence most of the cultured cells had a polarized-elongated appearance. Mature taste cells consist of several different histological subtypes and exhibit taste cell specific features. Here we show that cells within these cultures display many molecular and physiological features characteristic of mature taste cells. Gustducin and phospholipase C – β2, (PLC-β2) mRNA were detected by reverse transcriptase-polymerase chain reaction and products confirmed by sequencing (Figure 2). Expression of gustducin and PLC-β2 was detected immunocytochemically indicating the presence of type II-like cells (Figure 3 B and D respectively). The prevalence and relative expression patterns of gustducin and PLC-β2 reflecting cell signaling pathways and cell types does not precisely reflect that seen in vivo. The factors governing the relative distribution of different cell types within a taste bud are unknown, and may not be replicated in the culture conditions. Therefore, both the similarities and differences between the cultured cells and their in vivo counterparts provide an opportunity to examine the regulation of these characteristics at a molecular level, under conditions which can be manipulated and monitored.

An important criterion for a model system is the presence of relevant functional properties. Cultured cells also exhibited robust increases in intracellular calcium in response to appropriate concentrations of several taste stimuli (Figure 4). In these studies, sweet and bitter stimuli elicit an increase in intracellular calcium that may correspond to a depolarization. These taste cell-like properties of the cultured cells lend support to the assertion that the model system we describe here will be a valuable asset for further studies of the transduction pathways and regenerative capacity of human taste cells.

Figure 1. Attachment and morphology of cultured human fungiform taste papillae cells. Primary human fungiform taste papillae cell cultures grown on collagen type-1 coated plates were imaged after 2 days (A). Human fungiform taste papillae cells grew for up to 4 weeks under attached cell clusters, which seemed to give rise to daughter cells. The cells typically grew to confluence within four weeks (B), and (C and D) represents day 2 and 4 weeks after harvesting and reseeding respectively. Cultured cells continued to grow for at least 8 months. Scale bars = 50 nm. Part of Figure 1 was also presented in Ozdener et. al.¹⁰.

Figure 2. RT-PCR results demonstrated the presence of specific taste cell biomarker mRNAs (gustducin and PLC-β2,). Total RNA from cultured human fungiform cells was reverse transcribed using the Superscript First Strand Synthesis System for RT-PCR (Invitrogen) and used for PCR by amplifying with specific primers designed for detection of gustducin and PLC-β2. Primers (Table 1) were chosen to span one or more introns. Each mRNA was detected in cultured human fungiform taste papillae cells from passage 4 or 5 with amplification products of the expected size, confirmed by sequencing. Specific mRNA was not detected in control experiments without reverse transcriptase indicating no genomic DNA contamination. PCR products were separated on 2% agarose gels and the amplified products on the gel were excised with a razor. DNA was extracted from the gel using QIAquick Gel Extraction Kit (Qiagen). PCR products were sequenced at the University of Pennsylvania Sequencing facilities. C-PCR and C-RT are control experiments for PCR and Reverse Transcriptase, respectively. Part of Figure 2 was also presented in Ozdener et. al.¹⁰.

Figure 3. Immunostaining of cultured human fungiform taste papillae cells passage 4 or 5 showed presence of taste cell biomarkers. Cultured human fungiform taste papillae cells (5 days old) were fixed with 4% paraformaldehyde, and incubated with primary antibodies (Table 2) overnight at 4 °C. Images were acquired with a Leica TCS-SP2 confocal laser scanning microscope. Approximately, 60% of cultured fungiform taste papillae cells were labeled with gustducin (B) and 20-30 % with PLC-β2 (D). Transmission images of corresponding cells are shown (A and C). For controls, immunostaining with antibody specific immunoglobulin demonstrated the absence of nonspecific immuno reactivity (data not shown). Scale bars = 50 nm. Part of Figure 3 was also presented in Ozdener et. al.¹⁰.

Figure 4. Cultured human fungiform taste papillae cells from passage 4 or 5 respond to different stimuli. Cultured human fungiform taste papillae cells (4-5 days old) were loaded with 1mM Fura-2 AM (Molecular Probes) and 10 mg/ml Pluronic F127 (Molecular Probes). Changes in intracellular calcium levels ([Ca²⁺]i) in cultured human fungiform taste papillae cells were measured using standard manual imaging techniques. The cells were visualized using an inverted fluorescence microscope at excitation wavelengths of 340 nm and 380 nm and an emission wavelength set by a band pass filter centered at 510 nm. Stimuli were dissolved in Bath solution and then pH and osmolarity readjusted if needed. Cultured human fungiform taste papillae cells responded to sweet and bitter stimuli. Graphs illustrate representative responses of [Ca+2]i levels in individual cells during exposure to (A) Denatonium (2 mM), (B) Sucralose (1 mM). Each trace represents single individual cells.

Table 1. Primers and conditions used for detecting taste specific molecules.

Table 2. Antibodies used for detecting expression of specific molecules.