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 35oC 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 36oC in a humidified incubator containing 5% CO2. CRITICAL STEP Temperature of the incubator should be 36oC.
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 36oC,
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 36oC in a humidified incubator containing 5% CO2. CRITICAL STEP Temperature of the incubator should be 36oC.
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 36oC,
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 -80oC 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 37oC 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.