Purification and Characterization of Fat Taste Receptor-Positive Cells from Mouse Tongue Papillae

Summary

We describe the isolation and purification of lipid gustatory cells that express functional CD36 receptor in mouse tongue papillae.

Abstract

Sweet, umami, bitter, salt, and sour are the five taste modalities; however, there is increasing evidence of a sixth taste modality related to the oro-sensory perception of dietary fatty acids. Fat taste is principally detected by cluster of differentiation 36 (CD36), G-protein-coupled receptor 120 (GPR120), and GPR40. Despite the high level of interest, it is very difficult to obtain ethical approval to isolate human taste bud cells (TBCs). Therefore, mouse TBCs are much sought after for in vitro studies. This study aimed to develop a method for the purification of CD36-expressing TBCs from mouse fungiform and circumvallate papillae.

After cervical dislocation, the tongue was removed, and an elastase/dispase enzyme mixture was injected under the epithelium and around the circumvallate papillae. The epithelium-containing taste buds were picked off and subjected to enzymatic digestion with the elastase and dispase mixture. The cells were isolated by using an anti-CD36 antibody coupled to phycoerythrin (PE) and anti-PE-antibodies coupled to magnetic beads. The mixture was then passed through a magnetic column in which the CD36-positive cells were retained.

The isolated cells were cultured for up to 5 days, and western blotting and quantitative reverse-transcription polymerase chain reaction (RT-qPCR) techniques revealed that purified cells expressed the receptors for CD36 and GPR120 as well as α-gustducin and phospholipase C (PLC) involved in downstream signal transduction. Using Fura-2-acetoxymethyl ester (Fura-2/AM), the selected positive cells were found to respond to dietary fatty acids via a CD36-induced increase in free intracellular Ca²⁺ concentrations. In conclusion, purified CD36-positive taste bud cells can be of great help for in vitro investigation of taste bud physiology and for studying the mechanisms of fat taste perception.

Introduction

Fat taste represents the sixth taste quality in addition to the five basic taste qualities, i.e., sweet, sour, bitter, salt, and umami^1,2. Taste buds, which are responsible for the gustatory perception of tastants, are mainly present in three lingual papillae, i.e., fungiform, foliate, and circumvallate. Taste buds consist of 4 types of TBCs with distinct functions: Type I (glial-like) cells, Type II (taste receptor) cells, Type III (neuronal-like) cells, and Type IV (progenitor) cells. Type II cells express the taste receptors for sweet, umami, fat, and bitter. Bitter, umami, and sweet tastes are detected by the type 2 taste receptor (T2R) and the heterodimers, T1R1/T1R3 and T1R2/T1R3, respectively. T1R and T2R are coupled to a G-protein called gustducin. Cluster of differentiation 36 (CD36) and two G-protein-coupled receptors (GPCRs), i.e., GPR120 and GPR40, are implicated in the gustatory perception of dietary fats in rodents³.

It is noteworthy that CD36 exhibits high affinity (in the order of nanomolar) for fatty acids⁴. Several reports have documented the expression of CD36 in lingual gustatory cells in humans⁵ and other mammals^6,7,8,9. As it is very difficult to obtain human TBCs, mouse TBCs must be isolated for in vitro studies. Hence, this study aimed to purify CD36-positive TBCs from enzymatically digested papillae by a positive selection approach using anti-CD36-PE and anti-PE-antibodies coupled to magnetic beads. This method gave greater purity of the selected cells with respect to their calcium signaling response when CD36 was activated by fatty acids. Thus, CD36-positive TBCs can be of great help to study the physiological aspects of fat taste signaling.

Protocol

NOTE: Male, 10-12-weeks-old C57BL/6J mice were used in this study. The general guidelines for the care and use of laboratory animals recommended by the Council of European Economic Communities were followed, and the protocol was approved by the Regional Ethical Committees "protocol number 16158". See Table 1 for recipes of media and buffers used in this protocol.

1. Tongue isolation

1. Sacrifice male C57BL/6J mice (n=10) by cervical dislocation.
2. Cut the side layers of the mouth up to the ears with scissors on both sides. Be careful not to cut the tongue. Hold the tongue with forceps, and cut the jaw ligament under the tongue and the bottom of the tongue.
   NOTE: Do not damage the circumvallate area; cut out additional tissues to avoid damage to the tongue.
3. Place the isolated tongue in a Petri dish with Iscove's modified Dulbecco medium (IMDM)/MCDB complete medium until the dissection of all the mice (~50 min). To get rid of blood and hair, wash the tongues in the same dish with IMDM/MCDB complete medium.

2. Isolation of lingual epithelium

1. Transfer the tongues to cold (4 °C) Tyrode solution with calcium and incubate for 5 min. Using a syringe with 26 G needle, inject ~200 µL of elastase/dispase enzyme mixture under the epithelium and around the circumvallate papillae. Incubate the injected tongues for 15 min at 37 °C in Ca²⁺-free Tyrode solution (in the CO₂ incubator).
2. Using scissors and forceps, peel off the epithelium containing fungiform papillae, remove the circumvallate papillae under a microscope, and place them in a microcentrifuge tube containing cold IMDM/MCDB complete medium (4 °C).

3. Isolation of taste bud cells

NOTE: Perform cell isolation in a laminar flow hood under sterile conditions.

1. Centrifuge the epithelium at 600 × g for 10 min at 4 °C, and discard the supernatant. Dissolve the pellet (epithelium) with 1 mL of the above enzyme mixture (step 2.1), cut the epithelium with scissors to facilitate enzyme action, and then incubate for ~10 min at 37 °C in the CO₂ incubator.
2. Transfer the supernatant into a new microcentrifuge tube, and perform a second round of digestion on the undigested tissue (debris) in the centrifuged tube from step 3.1. Centrifuge the tube containing the digested tissues at 600 × g (10 min, room temperature (RT)), remove the supernatant, suspend the pellet containing dissociated cells in the IMDM/MCDB complete medium, and keep the tube in the CO₂ incubator (37 °C, 5% CO_2, and 95% humidity) until the end of the isolation.
3. Repeat steps 3.1-3.3 three times with 1 mL of the enzyme mix to dissociate the undigested tissue. Pool all the dissociated cells into a 15 mL tube.

4. Purification of CD36-positive cells

NOTE: Magnetic separation of CD36-positive cells was performed according to the kit manufacturer's instructions (see the Table of Materials and Figure 1).

1. Remove cell clumps by passing the cells through 70 µm pre-separation filters to avoid clogging the column; count the number of cells. After centrifugation (300 × g/10 min), suspend the pellet in 1x magnetic-activated cell sorting (MACS) bovine serum albumin (BSA) buffer to obtain a concentration of 10⁷ cells/80 µL of the MACS buffer
2. Add anti-CD36-PE (20 µL/10⁷ cells), mix gently, and incubate for 10 min in the refrigerator (2−8 °C). Wash the cells by adding 2 mL (per 10⁷ cells) of MACS buffer, centrifuge (300 × g for 10 min) the cell suspension.
3. Suspend the pellet in 80 µL of MACS buffer (per 10⁷ total cells), and add 20 µL of anti-PE-coupled microBeads (per 10⁷ total cells). Mix gently and incubate for 15 min in the refrigerator (2−8 °C).
4. Wash the beads by adding 2 mL (per 10⁷ cells) of MACS buffer, followed by centrifugation (300 × g for 10 min). Resuspend up to 10⁷cells in 500 µL of MACS buffer.
5. Magnetic separation with a MS Column (Table of Materials)
   1. Place a MS column in the magnetic MACS Separator, and rinse it with 500 µL of MACS buffer. Apply the cell suspension onto the column, and wash the column three times with 500 µL of MACS buffer. Collect the flow-through containing unlabeled cells.
   2. For elution of the CD36-positive, labeled cells, remove the column from the separator and place it on a collection tube. Pipette 1 mL of MACS buffer onto the column, and immediately flush out the magnetically labeled cells by firmly pushing the plunger into the column.

5. Cell culture

1. Centrifuge the cells (300 × g, 10 min, RT), discard the supernatant, and suspend the pellet in IMDM/MCDB complete medium. Distribute the cell suspension into wells for culture up to 5 days in CO₂ incubator (37 °C, 5% CO₂, and 95% humidity).
   NOTE: The duration of the purification procedure for CD36-positive cells is ~8 h.

النتائج

After selection, all purified cells were found to co-express CD36 along with α-gustducin (Figure 2A). The expression of CD36 (Figure 2B) and α-gustducin (Figure 2C) was high compared to that of CD36-negative cells or cells before selection. Thus, these purified cells are fat taste receptor cells (type 2 cells). As CD36 represents the main sensor of dietary long-chain fatty acids (LCFAs) in taste buds, we investiga...

Discussion

CD36-positive taste bud cells were isolated from tongue fungiform and circumvallate papillae using a positive selection approach with anti-CD36 antibodies, which offers greater purity due to the specificity of the reaction in comparison to negative selection. All these cells express CD36 and are type II cells as they co-express α-gustducin. This was in accordance with previous work⁶ demonstrating colocalization of CD36 and α-gustducin by immunohistochemistry on a whole tongue and the res...

Materials

Name	Company	Catalog Number	Comments
amphotericin B (250 µg/mL)	PAA	P11-001
anti-CD36	Atlas Antibodies	HPA002018
anti-α-gustducin	Santa Cruz	sc-395
Anti-PE MicroBeads	Miltenyi Biotec	130-048-801
collagenase	Worthington Biochemical	LS004196
CD36 Antibody, anti-mouse, PE, REAfinity	Miltenyi Biotec	130-122-084
Dispase	Worthington Biochemical	LS02109
Elastase	Worthington Biochemical	LS02292
fetal calf serum (FCS)	Dominique Dutscher  500105EE	500105EE
gentamycin (10 mg/mL)	PAA Laboratories  P06-03050	P06-03050
Iscove's Modified Dulbecco's Medium (IMDM)	Pan biotech	P04-20150
MACS buffer BSA Stock Solution	Miltenyi Biotec	130-091-376
MCDB 153	Alphabioregen	PG053
MS Columns	Miltenyi Biotec	130-042-201
N-succinimidyl oleate ester (SSO)	Sigma	SML2148
OctoMACS Starting Kit	Miltenyi Biotec	130-042-108
penicillin/streptomycin (10,000 U/10,000 µg/mL)	Thermo Fisher  15140-122	15140-122
Pre-Separation Filters (70 µm)	Miltenyi Biotec	130-095-823
Trypsin inhibitor	Worthington Biochemical	LS0028292