Adult mice were housed in a temperature-controlled room (22°C) under artificial illumination on a regular 12-hour day/night cycle and 55% relative humidity with access to food and water ad libitum. The mice used in the study were adult (6-8 weeks±5 days) male and female animals.

Tongues from sacrificed adult mice were dissected and injected with ~0.5 mL of dispase II (2.2 unit/mL; Roche, Basel, Switzerland) in phosphate-buffered saline (PBS) for 25 minutes at 37°C. The tongue epithelium was detached gently from the underlying tongue mesenchyme. The epithelium of circumvallate and foliate papillae was dissected and incubated with TrypLE Express for 30 minutes at 37°C and centrifuged at 800 rpm for 20 minutes. The cell pellet was resuspended in Matrigel and seeded onto 24-well culture plates (50 μL of Matrigel). Matrigel was allowed to polymerize for at least 10 minutes at 37°C. A taste culture medium based on DMEM/F12 supplemented with N2 (1%), B27 (2% [vol/vol]), R-spondin-1 (200 ng/mL), noggin (100 ng/mL), jagged-1 (1 μM), Y27632 (10 μM), N-acetylcysteine (1 mM), and epidermal growth factor (50 ng/mL) was added to the plate. The growth medium was changed every 3 days. For passage culture, organoids were transferred into TrypLE solution and dissociated into small pieces mechanically using a fine glass pipette. The solution was then passed through a cell strainer to obtain single cells. Cells were pelleted by centrifuging at 600 × g for 5 minutes. Single cells were re-embedded into fresh Matrigel and plated in 24-well plates.

Single lingual epithelial cells were embedded and cultured in Matrigel for 4 days with growth medium, as mentioned above. The Matrigel-embedded organoids were washed with ice-cold basal medium to break down the Matrigel by inverting and subsequently incubating on ice for 20 minutes. The organoids were gently centrifuged at 900 rpm for 5 minutes at 4°C, and the supernatant was removed carefully using a Pasteur pipette without disturbing the pelleted organoids. Fresh ice-cold basal medium was added for washing. This process was repeated three times. The organoids were resuspended in growth medium and transferred to ultra-low attachment 96-well culture plates (Costar; Corning Inc., Corning, NY, USA). Suspended organoids were cultured at 37°C with 5% CO₂.

The organoids were fixed in 4% paraformaldehyde and processed until Optimal Cutting Temperature (OCT) compound using standard procedures. Sections (7 μm) were prepared for immunostaining. For immunostaining, the slides were boiled in citrate buffer (pH 6.0). Blocking was carried out using 1% goat serum or 5% bovine serum albumin in PBS. The slides were incubated with antibodies against GLUT1 (1:200), GLUT2 (1:200), IGF1R (1:200), NGFR (1:200), K8 (1:200), Ecad (1:200), pMAPK (1:100), and pMEK (1:100) at 4°C overnight. The following day slides were washed and sequentially incubated with a secondary antibody (1:200; Invitrogen, Waltham, MA, USA) and counterstained with DAPI to visualize nuclei. The sections were examined using a confocal laser microscope (Leica DMi8).

The total RNA was extracted using the TRIzol reagent (#15596-026; Thermo Fisher Scientific, Waltham, MA, USA). The extracts were reverse-transcribed using Maxime RT PreMix (#25081; iNtRON, Seongnam, Korea). Real-time quantitative polymerase chain reaction (RT-qPCR) was performed using a StepOnePlus Real-Time PCR System (Applied Biosystems, Waltham, MA, USA). The expression levels of each gene are expressed as normalized ratios against the B2m housekeeping gene.

Results were expressed as the mean±standard deviation. GraphPad Prism 7 (GraphPad Software, San Diego, CA, USA) was used to analyze the data. Comparisons of two groups were performed using the unpaired two-tailed t-test. A p-value <0.05 was considered significant.

To alter their apicobasal polarity, the organoids cultured in Matrigel were transferred to suspension culture in a low-attachment dish after 96 hours (Fig. 1A). To examine the altered morphology between the two culture methods, hematoxylin and eosin staining was performed. As reported in our previous study [6], a keratinized inner core was observed in the Matrigel-cultured organoids that was not seen in the suspension-cultured organoids (Fig. 1B and 1C). Furthermore, taste receptor cells expressing keratin 8 (K8) were localized inside the Matrigel-cultured organoids, but on the periphery in the suspension-cultured organoids, indicating alteration of apicobasal polarity after suspension culture (Fig. 1D and 1E). The NGFR p75 is a pan-neurotrophin receptor that binds to neurotrophins to promote neuronal development, survival, and differentiation [14]. In the Matrigel-cultured taste bud organoids, NGFR p75 expression was observed on the periphery of the organoids, and NGFR p75-positive cells were not observed inside the Matrigel organoids where mature taste receptor cells were localized (Fig. 1F). In contrast, localization of NGFR p75 was observed on the periphery of the taste bud organoids and inside the suspension-cultured organoids (Fig. 1G). These results indicate that the NGFR p75 was co-localized with K8-positive taste receptor cells in the suspension-cultured taste bud organoids.

Glucose transporter isoforms such as GLUT1 and GLUT2 are expressed by taste receptor cells [12,13]. To investigate whether taste bud organoids also expressed glucose transporters, immunohistochemistry was performed for GLUT1 and GLUT2 transporter channels with the epithelial cell marker E-cadherin. GLUT1 was observed to co-localize with epithelial cells both inside and on the periphery of Matrigel- and suspension-cultured organoids (Fig. 2A and 2D). GLUT2 staining co-localized with E-cadherin-expressing epithelial cells on the periphery of Matrigel-cultured organoids, whereas the suspension-cultured organoids showed co-localization both on the periphery and inside the organoids (Fig. 2B and 2E). NGFR p75 co-localized with E-cadherin-positive epithelial cells on the periphery of Matrigel-cultured organoids (Fig. 2C). In contrast, after suspension culture of taste bud organoids, NGFR p75 was observed both on the periphery and inside the organoids (Fig. 2F). IGF1R is expressed in the taste buds and interacts with insulin-like growth factors, which are secreted by the neural ganglia and are required for cell growth and differentiation [11]. Immunohistochemical staining revealed that IGF1R was not detected in the Matrigel-cultured taste bud organoids (Supplementary Fig. 1A). However, IGF1R was partially observed in the cells of the suspension-cultured organoids (Supplementary Fig. 1B). These results indicate that the taste epithelial cells in the suspension-cultured organoids expressed GLUT1 and GLUT2 like in vivo taste buds.

A subtype of type II taste receptor cells, belonging to the type I taste G protein-coupled receptor family, forms a dimer that functions as a sweet, bitter, or umami receptor. Sweet taste is mediated by the heteromeric complex of Tas1r2 (taste receptor type 1, member 2) and Tas1r3 (taste receptor type 1, member 3) [15,16]. After 14 days of culture, the expression level of sweet taste receptor genes was significantly higher in suspension-cultured organoids than in Matrigel-embedded organoids (Fig. 3A). Furthermore, to validate the immunohistochemistry data, which showed increased expression of growth factor receptors and glucose transporters after suspension culture, the expression levels of Ngfr and Glut2 were analyzed. Both Ngfr and Glut2 were upregulated in suspension-cultured organoids compared to Matrigel-embedded organoids (Fig. 3B). Thus, taste receptor cells, growth factor receptors, and glucose transporters were maintained and enhanced after suspension culture.

Previous studies have shown that NGFR and IGF1R signaling occurs via distinct receptor tyrosine kinases, which then activate common intracellular signaling pathways, such as Ras, mitogen-activated protein kinase (MAPK), phosphoinositide 3-kinase, and the serine-threonine kinase Akt [17]. To examine the activation state of these receptors in the taste bud organoids, immunostaining for intermediate signaling molecules was performed using phospho-specific antibodies. Immunostaining showed localization of phospho-MAPK and phospho-MEK in the nucleus of a subset of peripheral cells in the Matrigel-embedded organoids (Fig. 4A and 4B). In the suspension-cultured organoids, phospho-MAPK and phospho-MEK were observed in peripheral cells, as well as in the cells present inside the organoids (Fig. 4C and 4D). These data indicate that NGFR p75 and IGF1R are functionally active in taste bud organoids.