Method for culture and maintenance of long term primary human fungiform taste cells

doi:10.1038/protex.2017.065

Method Article

Method for culture and maintenance of long term primary human fungiform taste cells

https://doi.org/10.1038/protex.2017.065

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Taste cells are highly specialized epithelial cells with unique developmental and physiological characteristics. The ability to culture and maintain mammalian taste cells in vitro provides a useful model for molecular studies of the proliferation, differentiation and physiological function of human taste receptor cells. Here, we describe a simple, efficient and reproducible protocol for in vitro culture of human fungiform taste papillae (HBO) cells. While this protocol is optimized for human tissues, it is readily adapted for use with other species. Cultured fungiform taste cells can be passaged, frozen and re-plated, and have been maintained for up to a year without loss of viability and while retaining the ability to generate differentiated cells exhibiting many molecular properties of mature taste cells. This method will enable researchers to develop and characterize novel taste molecules and new therapeutic targets to promote regeneration following injury from surgery, radiation, atrophy, toxic exposures or deterioration due to aging or neurodegenerative disorders.

Cell biology

Biological techniques

Neuroscience

taste cells

Fungiform papillae

sweet

fat

bitter

umami

salt

Taste cells are highly specialized epithelial cells with unique histological, molecular and physiological characteristics that permit detection of a wide range of both structurally simple and structurally complex molecules. Taste receptor cells are organized in taste buds located within the taste papillae of the tongue. Recently, several studies have also reported the presence of taste cell/taste like cells and taste receptors in tissues such as the digestive system^1,2, respiratory system^3,4, brain⁵, testicles^6,7,8, pancreas^9,10, urinary bladder¹¹, liver⁹, and spermatozoa¹².

Early studies indicated that maintaining rat primary cells beyond 3 - 5 days was not possible based on the specified isolation procedures and culture techniques^13,14,15,16. Much of our current knowledge of taste cells has been uncovered through the use of freshly isolated primary cells, explant cultures, or nonhuman species to study taste cell development and physiological properties^17,18. These methods are time intensive and require the use of a large number of experimental animals. These methods are not conducive to screening large numbers of chemicals for taste activity. We first reported on a method to culture rodent taste cells¹⁹ and later reported the generation of cultures of human fungiform taste cells that continue to divide in culture for at least 10 cell passages^20,21. A variety of methods were used to show that cells within these cultures express proteins characteristic of mature functioning fungiform taste receptor cells. In addition, physiological responses to a variety of taste stimuli (bitter, fat, salt, umami and sweet) are observed in subsets of cells indicating functional maturation. Given the relative ease in applying the technique, and a great amount of progeny that can be generated, the taste cell culture method has been adopted in several laboratories. It has been used as a model for examining fat taste signaling pathway^22,23 and fat taste modulation²⁴ and also to study salt taste signaling and modulation²⁵. The taste cell culture protocol has been used to establish 3D taste cell culture²⁶ and in the development of a taste cell chip²⁷. This protocol has also been used as a model to study stem/progenitor cell cycle in taste bud organoid²⁸, and taste cell development²⁹ and in the development of a bitter blocker³⁰. It has also been adapted the studies of precursor porcine gustatory cells³¹. Through these collaborative and independent studies using this protocol, we have acquired an in-depth understanding of the variability, critical steps and most likely causes for difficulties, which are presented in this report in order to aid in further applications. The establishment of this human fungiform taste cell culture protocol has provided an important in vitro model to study intracellular signaling mechanisms, the impact of trophic or toxic agents on taste cell growth and function, and the assessment of chemical stimuli that may elicit taste responses and/or modulate responses to other stimuli. Taken together, the primary taste cell isolation and culture technique, as described herein, are relatively simple and rapid, yet allow for the direct comparison of normal human taste receptor physiology.

• Human fungiform taste papillae ! CAUTION Requires Institutional Review Board (IRB) approval of protocol and informed consent from volunteers. Biopsy procedures were performed by a licensed oral surgeon following appropriate safety and health requirements for experimentation with human tissue.

• Iscove’s Modified Dulbecco’s medium (stocks from different suppliers have been tested

and used with equivalent efficacy)

• MCDB 153 medium (Sigma)

• Fetal bovine serum (FBS; stocks from different suppliers have been tested and used with equivalent efficacy)

• Goat Serum (Vector Laboratories)

• Bovine serum albumin (BSA, Sigma)

• Penicillin-Streptomycin Fungizone liquid (10X, Invitrogen)

• Gentamicin (Sigma)

• Phosphate-buffered saline (PBS), pH 7.4, sterile

• 0.25% w/v trypsin-EDTA (Invitrogen)

• Rat tail collagen type 1 (BD Sciences, San Diego, CA)

• Dimethyl sulfoxide (DMSO, Sigma)

• Elastase (Sigma and Worthington)

• Collagenase (Worthington)

• Trypsin Inhibitor (Worthington)

• NaHCO3 (Sigma)

• NaH2PO4 (Sigma)

• Glucose (Sigma)

• NaCl (Sigma)

• KCl (Sigma)

• Sucrose (Sigma)

• MgCl2 (Sigma)

• CaCl2 (Sigma)

• Na-pyruvate (Sigma)

• Hepes (Sigma)

• EDTA (Sigma)

• NaOH (Fisher)

• Nitric acid (HNO3, Fisher)

• Ethanol (Fisher)

• Methanol (Fisher)

• Hydrogen Peroxide (Sigma)

• Trypan Blue (Sigma)

• Tris pH 7.2, (Fisher)

• Goat anti-T2R47 antibody (L-22; sc-395, Santa Cruz Biotechnology)

• Rabbit anti-PLC-β2 antibody (Q-15; sc-206, Santa Cruz Biotechnology)

• Rabbit anti-T1R2 antibody (LSBio)

• Rabbit anti-ENaC-δ antibody (LSBio)

• Rabbit anti-TRPM5 antibody (Alomone)

• QIAquick Gel Extraction Kit (Qiagen)

• Fura-2 AM (Molecular Probes)

• Pluronic F127 (10 mg/ml Molecular Probes)

• Small fine-tip forceps and scalpel blade

• Micro Iris scissors curved, sterile

• T-75 tissue culture treated flasks

• T-25 tissue culture treated flasks

• 12-well tissue culture treated dish

• Refrigerated tabletop centrifuge with swinging bucket rotor and adaptors for

• 15 ml conical capped tubes, sterile

• Certified laminar flow hood

• Cell culture incubators (36^oC, humidified, 95% air 5% CO₂)

• Ultralow-temperature storage space (-80^oC)

• Light microscope

REAGENT SETUP

Human fungiform taste bud Collect fungiform papillae from the dorsal surface of the anterior portion of the tongue using curved spring microscissors and place immediately into an isolation solution. This protocol is optimized for use with human tissue samples but can be applied to tissues from other species as well. Typically, six – eight papillae from each of three individuals are pooled to initiate a starter culture³².

MCDB 153 medium Dissolve a package of MCDB 153 (17.6 gram) completely in one liter of tissue culture grade water and supplement it with 1.18 g/L sodium bicarbonate accordingly manufacturer instructions and the check pH (7 .0 – 7.1). Store at 4⁰C and protect from light CRITICAL MCDB 153 medium is light sensitive and has a short shelf life (max 6 months after preparation). Cover medium bottle with aluminum foil. It should be colorless after preparation; any change of color is an indication of expiration.

Taste cell culture medium Iscove’s Modified Dulbecco’s medium (Gibco BRL, New York, NY) containing 10% fetal bovine serum ((FBS) BTI, Stoughton, MA), 1:5 ratio of MCDB 153 (Sigma) and a triple cocktail of antibiotics (100U/ml / 100μg/ml, Penicillin/Streptomycin, 2.5μg/ml Gentamycin and 0.5μg/ml Fungizone). CRITICAL Medium is light sensitive, cover with aluminum foil and use in 3 months or less.

Taste cell isolation solution Dissolve 2.1 g NaHCO3, 0.3 g NaH2PO4, 3.6 g glucose, 3.77 g NaCl, 1.48 g KCl, 0.372 g EDTA in nuclease free water and filter sterilize

Preparation of coverslips: OPTIONAL Prior to using, treat coverslips with 2M NaOH for 1 h and leave overnight in 70% nitric acid (HNO3). Wash with 9M HCl acid for 1 hour and autoclave coverslips in water and rinse with 70% ethanol and 100 % ethanol, and then air dry.

Coating coverslips with rat tail collagen type -1 Dilute rat tail collagen type-1 (3.96mg/ml,) with sterile nuclease-free water at 1:4 ratio. Add 0.5 -1 ml of rat tail collagen type-1 onto coverslips into 12 wells tissue culture plate for 15 minutes at room temperature. Remove rat tail collagen type-1 and let coverslip air dry for 10-20 minutes. ATTENTION Perform this step in sterile cell culture hood.

Enzyme mixture Mix collagenase (550 U/ml) + elastase (10 U/ml) and Trypsin inhibitor (0.9 mg/ml) in calcium-free Ringer solution just before use. CRITICAL Use the highest concentration of enzyme stock possible to minimize volume needed. Enzyme loses its activity under inappropriate storage and use.

Ringer solution (MHNK, pH 7.1-7.2, 300-310 mOsmol) Dissolve 145 g NaCl, 0.373g KCl, 0.203g MgCl2, 0.147g CaCl2, 0.110g Na-pyruvate, 4.76g Hepes-Na in 1 liter water and adjust pH to 7.1 to 7.2 and osmolarity to 300-310

Calcium free Ringer solution (MHNK w/o Ca+2, pH 7.1-7.2, 300-310 mOsmol) Dissolve 145 g NaCl, 0.373g KCl, 0.203g MgCl2, 0.110g Na-pyruvate, and 4.76g Hepes-Na and 2mM EDTA in 1 liter water and adjust pH to 7.1 to 7.2 and osmolarity to 300-310

Modified Ringer solution (pH 7.1-7.2, 300-310 mOsmol) Dissolve 4.67g NaCl, 0.373g KCl, 0.203g MgCl2, 0.147g CaCl2, 0.110g Na-pyruvate, and 4.76g Hepes-Na in 1 liter water and adjust pH to 7.1 to 7.2 and osmolarity to 300-310 by 5M NaCl. Filter sterilization.

Freezing medium Add DMSO into Fetal Bovine Serum (FBS) to make final volume 5% CRITICAL DMSO may use its physiologic properties when it gets oxidized therefore keep tightly closed and replace frequently.

PROCEDURE (Figure 1)

1- Obtaining human fungiform taste papillae • TIMING 3-4 minutes/individual

(i) Remove four to eight human fungiform taste papillae from the dorsal surface of the anterior portion of the tongue using sterile curved spring microscissors and sterile glassware and tubing

(ii) Place immediately into an isolation solution³²

2- Human fungiform taste papillae digestion • TIMING 30-45 minutes

Human fungiform taste cell papillae can be dissociated using enzymatic digestion protocol.

(i) Incubate fungiform papillae CRITICAL STEP with collagenase, elastase and soybean trypsin inhibitor in 35^oC water bath with circulation for 30 minutes and gentle oxygenation with 95% O₂/5% CO₂. CRITICAL STEP. After incubation, wash papillae with ringer and triturate the papillae 10 times with fire-polished glass Pasteur pipette for 10 times¹⁴

3- Centrifuge for 3 minutes at 2500 rpm at RT; remove isolation solution and add 1 ml of taste cell culture medium

4- Transfer digested fungiform papillae into glass dish

5- Finely mince fungiform papillae gently with surgical razor CRITICAL STEP

6- Add 150-250 μl of minced papillae into cloning cylinder onto rat tail collagen type-1 coated coverslip. TROUBLESHOOTING

7- Add 1 ml of taste cell culture medium into each well

Culturing of human fungiform taste cells • TIMING 3-4 weeks

8- Incubate plate at 36^oC in a humidified incubator containing 5% CO₂. CRITICAL STEP Temperature of the incubator should be 36^oC.

9- Place in an incubator undisturbed for 2-3 days prior to the first change of complete medium. Taste cells will eventually bind to the coated coverslip, although this may not be clearly visible in 1 to 5 days (Figure 2 A). You may observe some cubical cells. These are distinct from the majority of cells. They do not show any sign of proliferation (Not shown). These cells will be removed after the first passaging (Figure 2 C). CRITICAL STEP The enzymes and taste cell medium described above does not support the growth of cellular contaminants. TROUBLESHOOTING

10- Remove cloning cylinder from plate and medium completely and add 1 ml of taste cell medium into each well.

11- Check cell growth under the microscope every other day. Most of the cells grow under cell clusters (Figure 2 A and B) Cell clusters usually detach after 2-3 weeks in culture.

12- Replace 1/3 of medium every 6-7 days CRITICAL STEP Do not change medium often and/or completely. TROUBLESHOOTING

13- Once 80-100% of the cloning cylinder is covered with cells (Figure 2 B), trypsinize cells using 0.25% w/v trypsin/EDTA for 2-3 minutes at 36^oC,

14- 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.

15- Remove supernatant and resuspend cells with 1 ml of taste cell medium.

Propagation of human fungiform taste cells TIMING 3-4 weeks

16- Transfer cells into the T25 plate and add 4 ml of taste cell medium (Figure 2 C). Maintain cells at 36^oC in a humidified incubator containing 5% CO₂. CRITICAL STEP Temperature of the incubator should be 36^oC.

17- Replace 1/3 of medium every 6-7 days until cultured taste cells have reached 100% confluence (Figure 2 D). At this time, to enable taste cells to passage, wash cells once with sterile PBS then trypsinize cells using 0.25% w/v trypsin/EDTA for 2-3 minutes at 36^oC,

18- After centrifugation as described above, resuspend in complete taste cell medium and transfer cells to fresh T-75 flasks (passage 1) • TIMING 3-4 weeks

19- Replace 1/3 of medium every 6-7 days CRITICAL STEP Do not change medium often and/or completely

20- Repeat steps 17 and 18 when cells have reached close to 100% confluence.

21- Split cells to no more than 1:4 dilution in a T75 flask to maintain the adequate growth of the cells over time. It should be noted that primary human fungiform taste cells have a reduced growth rate as compared with immortalized cell lines. At this point, you may proceed with freezing vials of passage-1 cells for archival purposes.

Freezing and thawing cultured human fungiform taste cells • TIMING 10-15 minutes

22- To freeze stocks of primary taste cells, after trypsinization, add complete taste cell medium and transfer cells to sterile 15 ml conical centrifuge tubes. Centrifuge at 2500 rpm for 5 min at room temperature.

23- Carefully discard the supernatant and gently resuspend cells with appropriate 1ml of freezing medium

24- Transfer cells to labeled, sterile cryovials, cap tightly, and place in a freezing container containing isopropanol. Place into a -80^oC freezer for at least one day prior to transferring indefinitely to liquid nitrogen. CRITICAL STEP A slow cooling rate may increase the viability of cells. Freeze as many passage-1 cells as possible. Higher passage number cells can be frozen as well.

25- To thaw a vial of frozen primary human fungiform taste cells, place at 37^oC water bath until just thawed (approximately 1 minute) while working in a laminar-flow cell culture hood.

26- Transfer cells and freezing medium to a sterile 15 ml conical centrifuge tube and add 5 ml of taste cell culture medium.

27- 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.

28- Continue to culture cells according to Step 16 to 25 until cells show evidence of senescence, based on viability, proliferation rate, and functional properties. Experiments are typically performed using cells at passage-2 through -7. However, the cell system may be used beyond that passage number.

TIMING

Step 1, Removing human fungiform taste papillae: 3-4 minutes/person (use 2-3 individuals)

Steps 2–7, Human fungiform taste papillae digestion, trituration, and dissection: 1 h

Steps 8-15, Culturing of human fungiform taste cells: 3-4 weeks

Step 16-21, Propagation of human fungiform taste cells: 3-4 weeks

Step 22-27, Freezing primary human fungiform taste cells: 10-15 minutes

TROUBLESHOOTING

Step 6 Processing and coating coverslip with rat tail collagen type -1 may result in no attachment or attachment of different type of cells

Step 9 We did not observe any cellular contamination by using enzymes and medium described above. However, if seen, problems are typically minimized by preparing fresh medium and enzymes.

Step 12 Cultured human fungiform taste cells that are not growing well and appear to have atypical morphology can usually be rescued by replacing what is likely expired taste cell medium, specifically MCDB 153, with fresh taste cell medium.

During the last 10 years, at least 50 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 2-5 d after plating (Figure 2 A), cells grew for up to 2-3 weeks under attached cell clusters, occasionally generating daughter cells (Figure 2 B). From surgically removed 4-8 human fungiform papillae, initial isolates of primary human fungiform taste cells reach approximately 2000- 8000 cells in 3-4 weeks in culture. Expansion in T25 cell culture flasks (passage 0) for additional 3-4 weeks will yield a culture still free of epithelial cell contamination (Figure 2 C). Primary human fungiform taste cells can be further expanded in culture to yield upwards of at least a million cells by passage-1 in 3-4 weeks. Primary taste cells will continue to grow for more than a year (Figure 2 D).

We found that IMDM supplemented with 20% MCDB 153 medium and 10% FBS provided good cell maintenance and growth on collagen-coated tissue culture plates and coverslips. We observed that papillae incubated with enzymes mentioned above resulted in cells that, if maintained in the IMDM supplemented with MCDB 153 showed specific growth environment for the particular type of cells. Additionally, because fibroblasts efficiently adhere to polystyrene, using specially pre-treated and rat tail collagen type-1 coated coverslip may result in preventing potential contamination by fibroblasts.

The cells can withstand cryopreservation; however, renewed growth of the cryopreserved cells in culture was considerably faster than the growth of cultures directly from the biopsy. This result is consistent with each experiment and isolation. Theoretically, the majority of cells should be frozen as passage-1 cell stocks. The morphology of the cultured human fungiform taste cells in culture was similar to cultured chemosensory cells described previously^33,34. Most cultured human fungiform taste cells maintained their original compact appearance of cell bodies with or without one or more processes for up to 15-30 days. After reaching 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 was detected by reverse transcriptase-polymerase chain reaction; products were of the expected size and confirmed by sequencing (Figure 3). The cDNA transcribed from total RNA was amplified with intron-spanning specific primers. Specific mRNA was not detected in control experiments without reverse transcriptase, indicating no genomic DNA contamination. Expression of gustducin and PLC-β2, TRPM5, T1R2, T2R47, and ENaC-δ was also detected immunocytochemically in HBO cells, though there is variation in the number of cells immunoreactive to specific antibodies, suggesting the presence of type II and type III-like cells (Figure 4 A and L). 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 5 A-C). In these studies, sweet (Sucralose), bitter (Denatonium), salt (Sodium Chloride), umami (Monopotassium glutamate, MPG), sour (Citric acid, CA) and fatty acid (Oleic acid) 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 can be a valuable asset for further studies of the transduction pathways and developmental processes of taste cells.

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Cultured human fungiform taste papilla cells was honorarily named as HBO cells after Hasan Basri Ozdener (Father of the author, MHO). We thank Aimee Myers, Esi Quayson, Bilal Malik and Nadia Elkaddi for technical skills and help.

A patent portfolio covering methods for culturing mammalian taste cells has been filed by Monell Chemical Senses Center and M.H.O. and N.E.R. are named as inventors. M.H.O. and N.E.R. receive research support and royalties. The other author A.I.S declares no competing interests.

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Method for culture and maintenance of long term primary human fungiform taste cells

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Method for culture and maintenance of long term primary human fungiform taste cells

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Abstract

Figures

Introduction

Reagents

Equipment

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Timing

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Anticipated Results

References

Acknowledgements

Additional Declarations

Associated Publications

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