Bio-artificial tongue with tongue extracellular matrix and primary taste cells

Accepted Manuscript Bio-artificial tongue with tongue extracellular matrix and primary taste cells Jung Seung Lee, Ann-Na Cho, Yoonhee Jin, Jin Kim, Suran Kim, Seung-Woo Cho PII:

S0142-9612(17)30654-3

DOI:

10.1016/j.biomaterials.2017.10.019

Reference:

JBMT 18301

To appear in:

Biomaterials

Received Date: 26 March 2017 Revised Date:

8 October 2017

Accepted Date: 8 October 2017

Please cite this article as: Lee JS, Cho A-N, Jin Y, Kim J, Kim S, Cho S-W, Bio-artificial tongue with tongue extracellular matrix and primary taste cells, Biomaterials (2017), doi: 10.1016/ j.biomaterials.2017.10.019. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT Bio-Artificial Tongue with Tongue Extracellular Matrix and Primary Taste Cells

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Jung Seung Lee†, Ann-Na Cho†, Yoonhee Jin, Jin Kim, Suran Kim, and Seung-Woo Cho*

Department of Biotechnology, Yonsei University, Seoul 03722, Republic of Korea

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*Corresponding author Prof. Seung-Woo Cho

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Department of Biotechnology, Yonsei University, Seoul 03722, Republic of Korea. Tel: +82-2-2123-5662; Fax: +82-2-362-7265; E-mail: [email protected]

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These authors contributed equally to this work.

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†

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Abstract

Artificial taste devices for tastant sensing and taste information standardization are attracting

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increasing attention with the exponential growth of the food and beverage industries. Despite recent developments in artificial taste sensors incorporating polymers, lipid membranes, and synthetic vesicles, current devices have limited functionality and sensitivity, and are complex

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to manufacture. Moreover, such synthetic systems cannot simulate the taste signal

transmissions that are critical for complicated taste perception. The current document

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describes a primary taste cell-based artificial tongue that can mimic taste sensing. To maintain viable and functional taste cells required for in vitro tastant sensing, a tongue extracellular matrix (TEM) prepared by decellularization of tongue tissue was applied to twoand three-dimensional taste cell cultures. The TEM-based system recreates the tongue’s

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microenvironment and significantly improves the functionality of taste cells for sensing

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tastant molecules by enhancing cellular adhesion and gustatory gene expression compared with conventional collagen-based systems. The TEM-based platform simulates signal

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transmission from tastant-treated taste cells to adjacent neuronal cells, which was impossible with previous artificial taste sensors. The artificial tongue device may provide highly efficient,

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functional sensors for tastant detection and in vitro organ models that mimic the tongue allowing elucidation of the mechanisms of taste.

Keywords: bio-artificial tongue device, tongue extracellular matrix, decellularization, primary taste cell, tastant sensing

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1. Introduction

The rapid growth of the food and beverage industries worldwide has prompted the

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development of artificial taste sensor systems that can mimic and replace the physiological functions of the human tongue, i.e., sensing and discriminating between various tastes

through the perception of tastant molecules. Taste-sensing mechanisms are not yet fully

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understood, and seem to be affected by numerous factors such as olfactory sense and the textures of tastant molecules. The human tongue can distinguish between five tastes—

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sweetness, sourness, bitterness, saltiness, and umami—through the perception of the tastereceptor cells (TRCs) in the taste buds, and the subsequent complex intracellular signaling cascades responsible for detecting specific molecules [1, 2]. The ability to recognize and precisely distinguish between tastant molecules is indispensable for quality control and

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standardization in taste inspection during the development and production of foods and

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beverages. However, to date this has been primarily dependent on human inspectors, which is time-consuming, is subject to significant variation among human subjects, and hinders the

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objective, quantitative analysis of taste perception [3, 4]. Over the last few decades, assay platforms have been concurrently developed with

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nanotechnologies and analysis techniques to overcome the current limitations in artificial taste sensing [4-10]. Indeed, various calorimetric assays incorporating lipid membranes or synthetic polymers combined with analytical tools have been proposed for artificial taste sensors [5, 6, 11, 12]. However, most of the systems developed to date do not respond simultaneously to multiple tastant molecules, and do not reflect complex taste signaling pathways. Moreover, their fabrication is usually complicated and requires analytical devices [4, 9]. Several studies have introduced proteins or vesicles with taste receptors in the assay platform to mimic more closely the role of TRCs in taste sensing. Recombinant human taste 3

ACCEPTED MANUSCRIPT receptor proteins and electronic nanomaterials have been integrated into artificial taste sensor platforms, and the developed systems enable the efficient detection of various tastant molecules with femto-molar levels of sensitivity [4, 8, 13]. Although these systems have

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exhibited improved taste-sensing performance in terms of sensitivity and accuracy, they do not achieve the unique functionalities of taste cells because they cannot simulate the complex and interconnected downstream signaling cascades involved in taste sensing. Moreover,

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further improvements to the stability of the membranous proteins extracted from cell membranes are required to ensure prolonged taste sensing [14].

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For these reasons, cell-based taste devices have recently been developed to mimic the physiological functions of the human tongue for accurate and delicate taste sensing. There have been several attempts to construct an artificial tongue using taste cells that respond to a wide range of taste compounds [15-17]. For example, Zhang et al. reported an in vitro

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gustatory system for recording the extracellular action potential of taste bud cells using a

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light-based sensor system [15]. Furthermore, cells expressing specific taste receptors have also been used for a cell-based artificial gustatory system for studying taste-sensing

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mechanisms [16, 17]. Despite the improved taste sensing performance of these cell-based systems, the culture and maintenance of taste cells with functional phenotypes is regarded as

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one of the critical hurdles in cell-based sensing systems. Substances facilitating cell adhesion such as extracellular matrix (ECM) components (e.g., collagen, fibronectin) and chemicals (e.g., poly-L-lysine (PLL), poly-L-ornithine) have usually been utilized to enhance taste cell attachment, viability, and functionality [18, 19]. However, owing to the insufficient functionality of single ECM components and the cytotoxicity of polymers, novel biomaterial substitutes for these conventional materials are urgently required for functional taste cell culture in taste-sensing systems.

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ACCEPTED MANUSCRIPT In this study, we developed a bio-artificial tongue platform with functional taste cells, enabling highly accurate and sensitive sensing of various tastes perceived by the human tongue: sweetness, bitterness, saltiness, and umami. Herein, we based bio-artificial tongue

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devices on two-dimensional (2D) and three-dimensional (3D) platforms composed of primary taste cells isolated from tongue tissue and decellularized tongue extracellular matrix (TEM). Because tissue-specific ECM can provide the tissue-specific microenvironments that are

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necessary for cellular activity and function [20-22], we have previously investigated the usefulness of decellularized matrix derived from various tissues for preparing 2D and 3D

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ECM platforms for tissue engineering applications [23, 24]. In this study, TEM-based 2D coatings and 3D hydrogel platforms prepared from decellularized porcine tongue matrix enabled the maintenance of functional taste cell-specific phenotypes. Moreover, the coatings and hydrogel platforms significantly improved the sensitivity of taste cells to various tastant

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molecules compared with collagen type I (Col I)-based platforms, which represent

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conventional taste cell culture systems. Interestingly, taste signal transmission between the tastant-treated taste cells and neurons, which is one of the most important events in taste

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perception, could be recreated in the TEM-based system. Finally, a taste cell-based sensor device was conceptually demonstrated in microfluidic devices with taste cells and TEM,

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emulating the tongue-specific microenvironments in functional taste cells. To the best of our knowledge, this is the first report of the development of bio-artificial tongue devices using primary taste cells and their niches for comprehensive taste sensing.

2. Materials and Methods

2.1. Taste cell isolation and culture

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ACCEPTED MANUSCRIPT The experimental protocol for the primary cell isolation from mouse tissues was reviewed and approved by the Yonsei Laboratory Animal Research Center (YLARC, IACUC-A201606-241-02). To obtain taste cells that would respond to tastants, the tongue was excised

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from a neonatal mouse (2 days old; C57BL/6; Nara Biotech, Pyeungtaek, Republic of Korea), sliced, and incubated in enzyme mixture (2 mL) comprising Accutase (1 mL; Gibco,

Gaithersburg, MD, USA) and Hank’s balanced salt solution (HBSS; 1 mL; Gibco) containing

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trypsin/ethylenediaminetetraacetic acid (Trypsin/EDTA; 0.025% (v/v); Gibco), 1  DNase I buffer (Gibco), and DNase I (8 U; Gibco) for 15 min at 37 °C. Subsequently, supernatant

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enzyme mixture was removed and the remaining enzyme mixture was inactivated by supplying trypsin inhibitor (2 mL; Sigma, St. Louis, MO, USA). The tongue tissues were then chopped and plated on a collagen type I (Col I; BD Biosciences, San Jose, CA, USA)coated cell culture plate, and incubated with Dulbecco’s modified Eagle’s medium: nutrient

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mixture F-12 (DMEM/F12; Gibco) containing 1  N2 supplement (Gibco), 0.5  B27

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supplement (Gibco), penicillin/streptomycin (1% (v/v); Gibco), and epidermal growth factor (EGF; 100 ng/mL; R&D Systems, Minneapolis, MN, USA) at 37 °C in a humidified 5% CO2

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atmosphere. To purify and increase the taste cell population, the migrated cells from the cultured tongue matrix were maintained under serum-deprived medium, as described above,

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and sub-cultured once for further application after 0.05% (v/v) trypsin/EDTA-mediated cell detachment process. For the in vitro experiments using 2D and 3D platforms, the taste cells were seeded onto the TEM-coated substrate for the 2D system and encapsulated in TEM hydrogel for the 3D system (Fig. 1A).

2.2. Flow cytometry analysis The population of taste cells used in our experiments was investigated using fluorescenceactivated cell sorting (FACS) analysis. Taste cells that had been sub-cultured once were 6

ACCEPTED MANUSCRIPT collected in tubes and washed three times with fetal bovine serum (FBS; 5% (v/v); Gibco) solution. Subsequently, the cells were stained using primary antibodies rabbit polyclonal IgG anti-gustducin (1:100; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and rabbit

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polyclonal IgG phospholipase C-β2 (PLC-β2, 1:100; Santa Cruz Biotechnology) diluted with flow cytometry staining buffer (R&D Systems). After 1 h of incubation, the cells were

washed with FBS solution (5%) and further incubated with secondary antibodies (goat anti-

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rabbit IgG H&L Alexa Fluor 488; 1:200; Invitrogen, Carlsbad, CA, USA) for 40 min. For the negative control group, the cells were incubated with the same secondary antibodies only.

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Finally, the cells were washed twice with FBS solution (5%) and resuspended in flow cytometry staining buffer; the cell population was then investigated using a FACSCalibur flow cytometer (BD Biosciences).

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2.3. Co-culture of taste cells and neurons

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To obtain primary mouse neurons, hippocampal neurons were isolated from embryos of ICR mouse at E14 (Orient Bio, Sungnam, Korea), as previously reported [25]. Briefly, the

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hippocampi were collected by dissecting microscopy, placed into a 15-mL tube containing Trypsin/EDTA (5 mL; 0.25%), and incubated for 15 min at 37 °C. Subsequently, the tissues

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were washed twice with 1  HBSS for 5 min at room temperature and resuspended in 1  HBSS (2 mL). The enzymatically digested tissues were dissociated by gentle pipetting to isolate the primary neurons. The isolated cells were then seeded onto TEM-coated (0.05 mg/mL) cell culture plates at a density of 1.5  104 cells/cm2 and cultured in neurobasal medium (Gibco) supplemented with 1  B27 (Gibco), 1  GlutaMAX (Gibco), and penicillin/streptomycin (1% (v/v); Gibco). For stable adhesion and culture of the isolated neurons, PLL (20 μg/mL, Sigma) was pre-coated and then laminin (2.5 μg/mL, Sigma) was coated with TEM. After 7 days of neuron culture, the taste cells were seeded at a density of 7

ACCEPTED MANUSCRIPT 3.0  104 cells/cm2 on the primary neuron-seeded surfaces. Two types of cells were cocultured using taste cell culture medium for 1 day after taste cell seeding and then analyzed

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for taste sensing and taste signal propagation.

2.4. Decellularization of tongue tissue

Porcine tongue was purchased from a regional market, sliced into 0.3-cm3 pieces, and stored

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in triple-distilled water (TDW) overnight before decellularization. The fully hydrated tongue tissue was then decellularized using detergent solutions as follows: sodium dodecyl sulfate

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(SDS; 1% (w/v); Sigma) for 24 h; Triton X-100 (1% (v/v); Wako, Osaka, Japan) with ammonium hydroxide (NH4OH; 0.1% (v/v); Sigma) for 2 h; TDW for 24 h (replaced every 6 h); and penicillin/streptomycin (1% (v/v); Gibco) for 1 h. The sterilized matrix was finally washed three times with TDW and lyophilized. All the decellularization processes were

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carried out at 4 °C at a stirring speed of 180 rpm. For 2D coating and 3D hydrogel formation,

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the decellularized TEM was solubilized at a concentration of 20 mg/mL using hydrochloric acid (HCl; 0.02 M; Sigma) and pepsin (4 mg/mL). Two different batches of porcine tongue

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tissue were decellularized and alternatively applied for preparing TEM-based coating or

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hydrogel throughout whole study.

2.5. Characterization of TEM For histological analysis, native and decellularized tongue matrices were fixed with paraformaldehyde (4% (v/v); Sigma), embedded in paraffin, and sliced into 6-μm-thick sections. Hematoxylin and eosin (H&E) staining was carried out to ensure the removal of cells and the preservation of ECMs after decellularization. The presence of Col I in the native or decellularized tongue matrix was confirmed by immunofluorescence staining with primary antibodies for Col I (1:100; Calbiochem, San Diego, CA, USA) and Alexa 594-conjugated 8

ACCEPTED MANUSCRIPT secondary antibodies (Invitrogen). Cells were counterstained with 4′,6-diamidino-2phenylindole (DAPI; TCI, Nihonbashi-honcho, Chuo-ku, Tokyo). The DNA content of the tissue was quantified using a DNA extraction kit (Bioneer, Daejeon, Republic of Korea). For

solution was used, as previously reported [23].

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2.6. 2D surface coating

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glycosaminoglycan (GAG) content analysis, 1,9-dimethyl methylene blue dye (Sigma)

The TEM solution was diluted with acetic acid (0.02 M; Sigma) and used to coat the surfaces

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of polystyrene cell culture plates. Col I was used as a control coating material. For surface coating, the cell culture plates were incubated with Col I (0.02 mg/mL), TEM (0.02 mg/mL), and TEM (0.1 mg/mL) for 1 h at 37 °C, and gently washed three times with phosphatebuffered saline (PBS, Sigma). For the in vitro experiments, the taste cells were seeded onto

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the TEM-coated substrates (48-well cell culture plate or 8-well cell culture chamber slide) at

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a density of 3.0  104 cells/cm2 for the 2D system. The surface atomic composition of the ECM-coated substrates was evaluated using X-ray photoelectron spectroscopy (XPS;

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ESCALAB 220i-XL; VG Thermo Scientific, East Grinstead, UK). The water contact angle was measured using a contact angle measurement system (Phoenix 300, Surface Electro

6).

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Optics Co., Suwon, Republic of Korea) after dropping TDW (10 μL) on each substrate (n =

2.7. 3D hydrogel formation To prepare the TEM hydrogel, TEM solution was mixed with 10  PBS, TDW, and sodium hydroxide (NaOH; 0.5 N; Sigma) solution. The final concentration of the PBS and the pH were adjusted to 1  and 7.4, respectively. For 3D cell culture in bulk TEM hydrogels, taste cell-containing pre-gel solution (1.0  105 cells/50 μl) was plated within a mold (inner 9

ACCEPTED MANUSCRIPT diameter = 5 mm, depth = 2.5 mm) and subsequently incubated at 37 °C for 30 min to generate fully crosslinked hydrogel. The internal structure of the hydrogel was confirmed by field emission scanning electron microscopy (FE-SEM; JSM-7001F; JEOL Tokyo, Japan).

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For SEM analysis, the hydrogel samples were serially dehydrated with ethanol (50, 60, 70, 80, 90, and 100%) and t-butyl alcohol (Sigma), and subsequently lyophilized. The rheometric analysis of the hydrogel was conducted using a rheometer (Bohlin Advanced Rheometer,

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Worcestershire, UK) in the frequency sweep mode. The storage modulus (G′) and the loss

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modulus (G′′) were measured at 1% strain from 0.1 to 1 Hz frequency.

2.8. Fabrication of microfluidic device for taste cell culture

The microfluidic devices were constructed from poly(dimethylsiloxane) (PDMS, Sylgard 184, Dow Corning, Midland, MI, USA) using a conventional soft-lithography process, as

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previously described [26]. The devices were fabricated to have three aligned microchannel

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structures with dimensions of 5 mm length, 1 mm width, and 150 μm height. To prepare the devices for taste cell culture, the PDMS constructs with microchannels and glass coverslips

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were autoclaved, exposed to ultraviolet light overnight, and then assembled after treating with oxygen plasma (CUTE; Femto Science, Seoul, Korea). For coating of the microchannels of

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the device, Col I (0.02 mg/mL) and TEM (0.02 and 0.1 mg/mL) solutions were applied to the device channel, incubated for 1 h at 37 °C, and gently washed three times with PBS (Sigma). Taste cells (6  104 cells resuspended in 18 μL of medium) were then seeded onto the central channel and allowed to adhere for 1 h. A sufficient amount of culture medium was added to medium chamber channels at both sides.

2.9. Evaluation of cell adhesion, proliferation, and viability

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ACCEPTED MANUSCRIPT For the observation of cell adhesion, the taste cells seeded on the substrates were fixed using paraformaldehyde (4% (v/v); Sigma) for 1 h and 24 h of culture. The cells were then stained using an Actin Cytoskeleton/Focal Adhesion immunofluorescence staining kit (Millipore,

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Temecula, CA, USA) following the manufacturer’s instructions, and the nuclei were counterstained with DAPI (TCI). The quantitative analysis of cell adhesion was carried out using ImageJ software (National Institutes of Health, Bethesda, MD, USA). The

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mitochondrial activity of the taste cells cultured on the substrates was quantified using a 3(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) assay (Sigma) at Days 1

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and 3. The viability of the taste cells cultured in the Col I and TEM hydrogels was measured using a Live/Dead cytotoxicity viability kit (Invitrogen) following the manufacturer’s protocol. Live cells (green) and dead cells (red) were visualized by fluorescence microscopy

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(1X 71, Olympus, Tokyo, Japan).

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2.10. Phenotypic analysis of taste cells

The phenotypic characteristics of the taste cells cultured on the substrates for 3 days were

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compared. For immunostaining, the cells were fixed with paraformaldehyde (4% (v/v); Sigma) and stained using the following primary antibodies: rabbit polyclonal IgG anti-

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gustducin (1:200; Santa Cruz Biotechnology) and rabbit polyclonal IgG PLC-β2 (1:200; Santa Cruz Biotechnology). The stained cells were visualized using Alexa-Fluor 488- and 594-conjugated secondary antibodies under a confocal microscope (LSM 880, Carl Zeiss, Oberkochen, Germany). DAPI (TCI) was used to counterstain the nuclei. The gene expression levels of the TRC markers were measured by quantitative real-time polymerase chain reaction (qPCR) with a TaqMan Gene Expression assay kit for the α-subunit of gustducin (GNAT3) (Mm01165313_m1; Applied Biosystems, Foster City, CA, USA), as previously described [27]. The relative gene expression of GNAT3 was normalized with that 11

ACCEPTED MANUSCRIPT of the endogenous reference gene, glyceraldehyde 3-phosphate dehydrogenase (GAPDH, Mm99999915_g1; Applied Biosystems) using the comparative Ct method.

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2.11. Calcium influx imaging For the functional testing of the cell-based taste sensor systems, calcium influx changes in taste cells as cellular responses to various tastant compounds were visualized with a

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fluorescent calcium indicator, Fluo-4-AM (Invitrogen) [28]. Taste cells cultured on Col I- or TEM-based platforms for 3 days were analyzed for calcium influx imaging. The cells were

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washed with 1  HBSS (Gibco) to remove medium components, and then labeled using a Fluo-4-AM calcium imaging kit (Invitrogen) by incubating them at 37 °C for 30 min and subsequently at room temperature for another 30 min. The cells were then further washed to remove the Fluo-4-AM-containing solution using 1  HBSS (Gibco). Each tastant solution

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(sodium chloride (NaCl; Sigma), sucrose (Sigma), calcium chloride (CaCl2; Sigma), glycine

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(Sigma), saccharin (Sigma), denatonium benzoate (Sigma), coffee powder (Kanu, Dong-Suh, Seoul, Korea), and wine (Soldepeňas, Felix Solis, La Mancha, Spain)) was added dropwise to

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the cell culture medium. Time-course images of the calcium influx changes were recorded using a confocal microscope (LSM 880, Carl Zeiss). The intensity of the signals was

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quantified by picking representative cells at the region of interest (ROI) and using ZEN software (Carl Zeiss). For calculating the relative intensity of signals (F/F0), the intensity at the ROI was normalized by the intensity at 0 s. The responsive population after tastant treatment was determined by counting the number of cells showing over 1.3-fold the intensity at 0 s.

2.12. Statistical analysis

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ACCEPTED MANUSCRIPT Statistical significance among the groups was determined by the Student’s t-test using GraphPad Prism 6 software (GraphPad Software, San Diego, CA, USA). Data were

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considered statistically significant at p ≤ 0.01 and p ≤ 0.05.

3. Results and Discussion

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3.1. Preparation of TEM-based 2D surface for taste cell culture

We applied decellularized TEM to the surface of a cell culture plate to prepare a TEM-based

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2D surface retaining the functional phenotypes of taste cells. Decellularized matrices with similar ECM compositions to those of native tissues enhance the viability, proliferation, and differentiation of cells and improve cellular functions [20, 23]. Because tongue is mainly composed of skeletal muscle ECMs including collagen, proteoglycan (PG), and GAG, the

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ECMs contained in decellularized TEM may be able to significantly influence the phenotypes

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and physiological functions of taste cells for taste sensing [29-32]. To reconstitute the biochemical and biophysical microenvironments of native tongue as taste cell niches,

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decellularized tongue tissue was processed into solubilized components and applied as a 2D surface coating material for taste cell culture (Fig. 1A).

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As the first step, porcine tongue was retrieved and decellularized serially using ionic

(SDS) and non-ionic (Triton-X 100 with NH4OH) detergent solutions (Fig. 1B, left panel). The decellularization process removed the cellular components but preserved the ECMs in the generated tongue matrix, as confirmed by H&E staining (Fig. 1B, middle panel). Immunohistochemical analysis revealed that a large amount of Col I remained in the decellularized tongue matrix (Fig. 1B, right panel). Complete removal of cellular components is essential in preparing decellularized matrix from native tissue, as the remnants of cellular components are directly related to immune reactions. In addition, GAG such as chondroitin 13

ACCEPTED MANUSCRIPT and heparan sulfate is known to provide privileged environment in maintaining structural and physiological integrity as well as gustatory sensing of taste buds [33]. Quantification of DNA and GAG contents further demonstrated that most cells had been removed (approximately 98%

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compared with native tongue tissue), whereas substantial GAG components were retained even after decellularization process (before – 16.3 ± 4.3 μg/mg, after – 10.6 ± 2.6 μg/mg), indicating successful decellularization of the tongue tissue (Fig. 1C, D). The TEM was then

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lyophilized and solubilized by treatment with pepsin to form the 2D surface coating and the 3D hydrogel.

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Solubilized TEM has versatile applicability to taste sensing because it can be transformed into various matrix formats [23, 34]. A polystyrene cell culture substrate was coated with TEM solution (0.02 mg/mL; TEM (0.02) and 0.1 mg/mL; TEM (0.1)) in acidic conditions (0.02 M acetic acid) to prepare a 2D cell culture platform with tongue-specific

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extracellular components. A Col I coating (0.02 mg/mL) was used as a positive control

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throughout the experiments because Col I is usually more beneficial for the maintenance of taste cells than supporting cells, Matrigel, and chemicals [19]. After surface modification

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with TEM, the surface atomic composition of the substrates was determined by XPS. Although the TEM coating did not cause a specific peak shift in C1s, a newly confirmed peak

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near 400 eV attributable to N1s and a peak near 532 eV attributable to O1s indicated adsorption by organic compounds such as ECM proteins and biomolecules on the TEMcoated substrates (Fig. 1E). Furthermore, the relative intensity of the N1s peak increased in proportion to the increase in TEM concentration in the surface coating (Fig. 1E), indicating that ECM adsorption on the substrate is dependent on TEM concentration. The change in surface property of the substrates was also evaluated by measuring water contact angle after TEM coating, and we confirmed the increment in hydrophilicity on the TEM-coated substrates compared with the non-coated substrate (no coating, 49.5° ± 1.6; TEM (0.02), 32.3° 14

ACCEPTED MANUSCRIPT ± 1.3; TEM (0.1), 30.6° ± 1.9) due to the presence of adsorbed proteins (Fig. 1F). To date, the challenge of stable cellular adhesion and retention of the unique performance of taste cells has hindered the development of effective cell-based platforms for artificial taste sensors and

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basic research gustatory models for taste sensing mechanism studies [18]. Surface engineering with tongue-specific ECM components would be expected to facilitate taste cell adhesion and improve taste cell-specific functions, in comparison with surface modification

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based on single ECMs or chemical components [23].

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3.2. Enhanced adhesion and functional phenotypes of taste cells on 2D TEM platform TEM surface coating significantly promotes taste cell adhesion, which is critical for taste sensing. The adhesion of taste cells to the TEM-coated substrates was examined by immunofluorescence staining of filamentous actin (F-actin) and vinculin for cytoskeletal and

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focal adhesion proteins, respectively. The taste cells were roughly purified from cell

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populations primarily isolated from the tongue tissue of neonatal mice through subculture in serum-depleted medium conditions. Flow cytometric analysis confirmed that the cell

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populations prepared by our method contained cells expressing gustducin (62.9%) and PLCβ2 (74.5%), typical phenotypic markers of TRCs (Fig. S1). Gustducin is one of the G-

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protein-associated taste receptors that play a crucial role in intracellular signal transduction for taste perception [35-37]. PLC-β2, an enzyme that is activated by G-proteins and calcium, is also involved in taste signaling pathways [35]. Therefore, cell populations exhibiting considerable gustducin and PLC-β2 expression are thought to be capable of differentiating and maturing into a native tongue gustatory system. After 1 h and 24 h of cell seeding, the initial adhesion and the spreading of the taste cells were greatly enhanced in the TEM groups (0.02 and 0.1) compared with the Col I group (Fig. 2A, B). Given that cellular adhesion is a critical event for phenotypic and functional maintenance of taste cells during long-term 15

ACCEPTED MANUSCRIPT culture [19], TEM-based 2D coating should be able to provide a favorable environment for a taste cell-based artificial sensor system. There was no significant difference in the proliferation of taste cells among the groups during 3 days of culture, as quantified by a MTT

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assay (Fig. 2C). Taste cell-specific phenotypes were readily retained on the TEM-based 2D coating platform. After 3 days of culture on each substrate, the taste cells on the TEM-coated

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substrates expressed much higher levels of gustducin and PLC-β2 proteins compared with the cells on the Col I-coated substrate, especially when the coating concentration of TEM was

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0.02 mg/mL (Fig. 2D). A qPCR assay indicated that gene expression of the GNAT3 in the taste cells was also substantially increased by TEM surface coating (Fig. 2E). Interestingly, we confirmed that TEM at a lower concentration (0.02 mg/mL) further promoted the adherence and phenotypic expression of taste cells. Thus, we speculate the potential utility of

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TEM coating for taste cell-based artificial tongue platforms.

3.3. 2D TEM-based artificial tongue for highly efficient and sensitive taste sensing

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Taste sensing in a 2D TEM-based artificial tongue with taste cells was evaluated by the analysis of the electrochemical responses of taste cells to various tastants. In this study, we

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examined the taste-sensing capacity of TEM-based artificial tongue platforms by quantifying the calcium influx of tastant-responding cells in the systems. The binding of soluble tastant molecules to taste receptors on TRCs in taste buds induces calcium channel activation and an increase in Ca2+ influx, initiating taste signaling cascades [38, 39]. Therefore, we investigated the changes in intracellular Ca2+ concentration to compare the levels of taste cell response to various types of tastant. To this end, calcium influx visualized by a calcium indicator, Fluo-4, was recorded after treating taste cells cultured for 3 days on either a TEM-coated or a Col I-

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ACCEPTED MANUSCRIPT coated substrate with gustatory molecules (Fig. 3) [40]. We tested various taste responses with NaCl (saltiness), sucrose (sweetness), saccharin (bitterness) [41], and glycine (umami). TEM coating improved the sensitivity and electrochemical functions of taste cells

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responding to a salty tastant. The precise perception of saltiness is important in the food and healthcare industries owing to the close relationship between salt consumption and the risk of developing adult diseases such as high blood pressure and cardiovascular malfunction [42].

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When taste cells were treated with NaCl solution (100 mM), the cells responded immediately, and there was a significantly higher level of calcium influx in the TEM groups (TEM (0.02)

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and TEM (0.1)) than in the Col I group, indicating increased sensitivity of the taste cells to saltiness following TEM coating (Fig. 3A, B). Furthermore, a higher portion of cells responded to NaCl in the TEM groups than in the Col I-coated group (Fig. 3C: Col I, 32.1 ± 15.1%; TEM (0.02), 72.0 ± 4.8%; and TEM (0.1), 62.2 ± 10.8%). Interestingly, when

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compared with the TEM (0.1) group, the cells in the TEM (0.02) group showed a

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significantly increased relative intensity of Ca2+ influx, demonstrating that there is an optimal concentration of TEM coating for substantial enhancement of taste sensing sensitivity.

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Next, we determined whether sweetness perception in the taste cells could also be improved using a TEM platform. Recently, sweet tastants have become more important,

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especially in the food and beverage industries, because sweet-tasting substances are enjoyable; however, this creates social and healthcare issues due to the increasing number of overweight and obese people [43]. Upon exposure to a sweet tastant (200 mM sucrose), taste cells on the TEM-coated substrates showed higher Fluo-4 intensity compared with those on the Col Icoated substrate (Fig. 3D, E). The percentage of sucrose-responsive cells also increased in the TEM groups, implying enhanced sweetness perception in the taste cells arising from TEM coating (Fig. 3F: Col I, 45.0 ± 17.3%; TEM (0.02), 94.1 ± 4.0%; and TEM (0.1), 93.8 ± 8.0%). There were no significant differences in the relative intensity of Ca2+ influx and the 17

ACCEPTED MANUSCRIPT responsive cell population between the TEM (0.02) and TEM (0.1) groups. The ability to perceive bitter compounds has developed in humans and animal species as a means of protection against toxins and harmful compounds [44]. Accordingly, bitterness

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detection can be useful for managing the freshness of food products and identifying rotten food. Saccharin has been widely used as an artificial sweetener, but it can also be used to test bitterness perception [41]. When saccharin (100 μM) was used to treat taste cells, the relative

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Ca2+ influx intensity sharply increased only in the TEM groups, whereas there was no

striking elevation of intensity in the Col I group (Fig. 3G, H). Although a similar percentage

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of cells in the two TEM groups responded to saccharin (Fig. 3I: TEM (0.02), 46.7 ± 5.4%; and TEM (0.1), 41.7 ± 10.0%), the cells cultured on TEM (0.02) generally showed a more dramatic improvement in susceptibility to the bitter tastant than those on TEM (0.1) (Fig. 3H). When CaCl2 was used as an alternative bitter tastant component, there was a significant

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increase in relative Ca2+ influx in the TEM (0.02) group (Fig. S2A, B). The percentage of

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CaCl2-responsive cells also increased significantly in the TEM groups (Fig. S2C), indicating TEM-mediated enhancement of bitterness perception and sensitivity in the taste cells.

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Umami tastants, which usually comprise amino acids and are highly attractive to humans owing to their enjoyable flavor [45], were efficiently detected with our TEM-based

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platform. When glycine (50 mM) was added to cells as an umami tastant, there was a steep elevation in the relative cytosolic Ca2+ concentration in cells in the TEM (0.02) group, whereas there was a relatively small increase of Ca2+ influx in the Col I and TEM (0.1) groups, indicating a highly sensitive response to umami tastants induced by TEM (0.02) coating (Fig. 3J, K). As with the other tastant stimulations, there was a higher percentage of glycine-responsive cells in the TEM groups than in the Col I group, although there was no statistical significance among the groups (Fig. 3L: Col I, 15.9 ± 6.0%; TEM (0.02), 28.0 ± 4.8%; and TEM (0.1), 23.3 ± 8.6%). 18

ACCEPTED MANUSCRIPT Overall, the results of our calcium influx-based taste sensing tests demonstrated the efficacy of the TEM-based taste sensor system. The system improved the taste cell-specific characteristics and responses to various tastant molecules without the need for purification of

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the taste-specific receptors or fabrication of complex devices. ECM proteins (e.g., fibronectin, laminin, tenascin) and certain growth factors (e.g., brain-derived neurotrophic factor, glial cell-derived neurotrophic factor) are crucial for the normal development and cellular function

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of the gustatory system [46, 47]. Therefore, a TEM-based platform, composed of tonguespecific ECMs and various bioactive molecules, can stably maintain the physiological

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functionality of taste cells and endow them with sensitivity to various tastants. A TEM-based artificial tongue could be used for the highly efficient sensing and detection of a wide variety of tastant molecules in the food industry and in healthcare management.

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3.4. Potential utility of TEM-based artificial tongue for commercial products

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To prove the potential utility of the TEM-based taste sensor system in terms of the requirements of the food industry, the cellular responses of taste cells to two commercial

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products (coffee and wine) were examined on the TEM-coated substrates. Coffee and wine are important drinks with enormous worldwide markets. In fact, because they generate

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complex tastes owing to the presence of various chemical compounds, the quality control of the products and the development of new products are quite complicated using current taste sensor systems [48]. Therefore, we determined the ability of the artificial tongue to efficiently sense bioactive compounds in coffee and wine. The relative reactivity of taste cells cultured on TEM-coated substrates with these two products was compared with that of the cells on the Col I-coated substrate. The cells grown on the TEM substrates exhibited more rapid responses to treatment with commercially available coffee powder (30 mg/mL) and higher calcium influx intensity than the cells on the Col I substrates (Fig. 4A, B), indicating that 19

ACCEPTED MANUSCRIPT TEM can markedly increase the cellular response and sensitivity to commercial products in taste cells. The portion of coffee-responsive cells was significantly greater in the TEM groups than in the Col I group (Fig. 4C). When diluted wine solution (1%, v/v), another commercial

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product, was used to treat the taste cells cultured on each substrate, the TEM coatings also induced increased calcium influx intensity and taste cell response to wine compounds (Fig. 4D-F). Thus, our results for coffee and wine demonstrated the potential utility of our TEM-

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and taste cell-based platform for sensing commercially available food products.

These calcium imaging-based functional tests confirm the improved sensitivity and

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functionality of taste cells following TEM surface modification; this probably results from providing the taste cells with similar biochemical and biophysical microenvironments to those found in native tongue tissue. However, the signal cascades involved in taste sensing are highly complex and large portions of the taste signaling process remain unknown [39].

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Therefore, the calcium mechanism alone may not fully explain the contribution made by

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TEM to the improved sensitivity, functionality, and phenotypic characteristics of taste cells. More comprehensive, extensive evaluations of our TEM- and taste cell-based artificial taste

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system are required. These systematic evaluations should involve a variety of tastant molecules, and should investigate the concentration-dependent responses of taste cells.

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Although electronic tongue (ET) and bioelectronic tongue (Bio-ET) have been rapidly

developed since last few decades with the advances in nanotechnologies and analysis techniques, taste cell-based artificial tongue systems are still advantageous in terms of mimicking complex and interconnected downstream signaling cascades involved in taste sensing, which cannot be recapitulated with electrical device- or protein-based sensor systems. However, to further increase commercial and practical utility of the TEM-based taste system in food and pharmaceutical analyses, convergence of our taste cell- and TEM-based system with emerging techniques such as bioelectronic devices that can record electronically cellular 20

ACCEPTED MANUSCRIPT signals with high accuracy and sensitivity and analytical tools of potentiometric and voltammetric types needs be considered, which would provide more objective and comprehensive interpretation of cellular responses to tastants.

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In this study, we examined changes in calcium influx to evaluate taste sensing functionality of cells after treating various tastants, but more specific initial process of taste sensing cascades could be investigated through modification of our taste system. In a taste

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sensing mechanism, different types of dimeric G-protein-coupled receptors (GPCRs) play pivotal roles in perception of various tastants. For example, sweet and umami tastants

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stimulate T1R1, T1R2, and T1R3, while bitter tastes are recognized through T2R-expressing cells [1]. Therefore, application of genetically modified taste cells (e.g., specific GPCRoverexpressing cells or fluorescence-expressing reporter taste cells when stimulated by specific GPCRs) as a cell source in our TEM-based system may be able to allow our system

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for the artificial sensor to examine the functional activity of dimeric GPCRs and provide

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basic information on the specific interactions between taste receptors and tastant molecules. Further studies to identify possible receptors could also be considered by using antagonists

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known to inhibit thermal and mechanosensitive responses in other sensory systems.

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3.5. Recreation of taste signal transmission between taste cells and neurons on TEM platform

The signal cascades of taste perception are initiated when tastant molecules bind to taste receptors on TRCs in the taste buds. Subsequently, neuronal and sensory cells surrounding the taste buds are activated and transmit the perceived signals to the brain through nerve fibers [1, 49]. Therefore, the interaction between the taste cells and the surrounding sensory cells such as gustatory neurons is as important as the interaction with the ECM environment [50]. To better understand the signal cascades involved in native gustatory sensing and the 21

ACCEPTED MANUSCRIPT perception process, such taste signal transmission needs to be realized in in vitro artificial systems. In this study, we attempted to reconstitute taste signal transmission by co-culturing

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taste cells and neurons on a TEM-based 2D platform, where neurons from hippocampus and taste cells served as signal recipients and donors, respectively. The taste cells were

fluorescently labeled with 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate

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(DiI) dye to distinguish them from responding neurons (Fig. 5A). During co-culture of taste cells and neurons, taste cell-mediated signal transduction to neurons was evaluated by

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determining the change of Ca2+ influx in the taste cells triggered by tastant treatment, and the subsequent propagation of calcium channel activation to neurons. When denatonium benzoate (5 μM), which simulates the bitter taste, was used to treat neurons in the absence of taste cells, none of the cells responded, indicating that hippocampal neurons do not respond

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directly to tastants without signal initiation by taste cells (Fig. S3 and Movie S1). However,

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when neurons were co-cultured with taste cells on the TEM-coated substrate, we clearly observed secondary responses in the neurons following primary activation of the taste cells

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by denatonium treatment, indicating taste signal transduction from taste cells to adjacent neurons (Fig. 5B, C and Movie S2). We could observe serial propagation of Ca2+ influx

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changes along with neurites from primary activation of taste cells (Fig. 5D). Together with the results from the culture of taste cells alone on TEM substrates, we

confirmed that TEM stably maintained and improved the functionality of taste cells, and established efficient interaction for signal transmission between taste cells and neuronal cells, which is essential for reconstituting complex taste perception processes. Importantly, previous artificial tongue devices using lipids, polymers, and various synthetic compounds have never achieved such signal transduction and cellular networks among taste cells and neuronal cells. Thus, our TEM-based artificial tongue platform may serve as a unique 22

ACCEPTED MANUSCRIPT gustatory model for basic research into the transformation of taste signals into electrochemical signals, and the interpretation of the perceived signals. Our data may indicate the possibility of innervation of sensory cells in the TEM-based system, but we did not

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measure the thermal and mechanosensitive receptors of innervated taste cells. Since TEM might be applicable for developing artificial sensory systems based on functional taste cells with innervation, we may need to check the thermal and mechanosensitive receptors

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innervated in the TEM-based systems in the future studies.

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3.6. 3D TEM hydrogel-based artificial tongue platform for enhanced sensitivity and functionality of taste cells

As the next step in the development of a highly functional taste device, TEM components were reconstituted into 3D hydrogel platforms, which were evaluated for efficient, sensitive

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taste sensing. 3D hydrogel systems using decellularized matrices from various organs and

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tissues have shown great potential in tissue engineering applications because they can generate tissue-specific microenvironments with the biochemical and biophysical cues that

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are essential for cellular function and survival [23, 51, 52]. To prepare a hydrogel platform for 3D taste cell culture, solubilized TEM crosslinking was induced by the temperature-

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mediated self-assembly of ECM components. Owing to the presence of large amounts of collagen components in the decellularized matrices, solubilized TEM forms a gel at 37 °C and neutral pH, which are the typical crosslinking conditions for collagen hydrogel formation [23]. TEM hydrogels of two different concentrations (5 and 10 mg/mL; 3D-TEM (5) and 3DTEM (10)) and Col I hydrogel (2 mg/mL; 3D-Col I) were prepared and characterized in terms of internal structures and rheological properties. As with the Col I-based hydrogels, the TEM hydrogels formed stable 3D constructs (Fig. 6A, insets) with collagenous nanofibrillar internal structures, as confirmed by SEM (Fig. 6A). Interestingly, perpendicular striations, a 23

ACCEPTED MANUSCRIPT typical characteristic of native collagen fibrillar structures, were observed in the nanofibrillar structures of the TEM hydrogels, as shown in the enlarged SEM images (Fig. 6A, bottom rows). Rheological analysis of the TEM hydrogels revealed the stable formation of 3D

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hydrogel constructs‚ which was confirmed by consistently high values of G′ (elastic modulus) than G′′ (viscous modulus) throughout all tested frequency ranges (0.1–1 Hz) (Fig. 6B). The 3D-TEM (5) hydrogel (19.2 ± 1.6 Pa) exhibited a similar average G′ to the 3D-Col I hydrogel

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(22.7 ± 4.3 Pa) at a frequency of 1 Hz (Fig. 6C). An increase of TEM concentration to 10 mg mL–1 led to an increased G′ in the formed hydrogel (3D-TEM (10), 58.6 ± 1.1 Pa) (Fig. 6C).

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When taste cells were cultured in the 3D-TEM (5) hydrogel, shrinkage occurred in the hydrogel construct. Therefore, the 3D-TEM (10) hydrogel with the higher G′ was used as the 3D culture platform for taste cell culture in further experiments.

The 3D TEM hydrogel platform significantly improved the viability, the functional

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phenotypic characteristics, and the sensitivity to tastant molecules of the taste cells.

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Following short-term culture of the taste cells in the 3D hydrogels (Days 0 and 1), more than 80% of the cells were viable in both hydrogel systems (3D-Col I, 83.6 ± 4.4% and 3D-TEM

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(10), 86.6 ± 1.7% at Day 0; 3D-Col I, 82.5 ± 1.4% and 3D-TEM (10), 84.1 ± 3.9% at Day 1) (Fig. 7A, B). However, the ratio of dead cells in the 3D-Col I hydrogel increased after 3 days

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of culture, and the viability was 71.2 ± 3.9% at Day 5, whereas taste cells encapsulated in the 3D-TEM (10) hydrogel remained highly viable during 5 days of culture (80.7 ± 3.2%) (Fig. 7A, B). Following 3 days of culture, taste cells cultured in the 3D-TEM (10) hydrogel exhibited much higher expression levels of the taste cell-specific markers gustducin and PLCβ2 than those in the 3D-Col I hydrogel (Fig. 7C, D), indicating the effectiveness of the TEM hydrogel for enhancing taste cell-specific characteristics. To confirm the ability of TEM hydrogels to enable the sensitive detection of tastant molecules, a salty tastant (200 mM NaCl) was used to treat the taste cells cultured in 3D TEM hydrogel, and the change in intracellular 24

ACCEPTED MANUSCRIPT Ca2+ concentration was determined by measuring calcium influx with Fluo-4 (Fig. 7E-G). A significantly stronger relative intensity of calcium influx was detected in the 3D-TEM (10) group compared with the 3D-Col I group (Fig. 7E, F). The quantification of taste cells

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responding to tastant molecules demonstrated that 86.0% (± 13.6) of the taste cells cultured in the 3D-TEM (10) hydrogel were stimulated by the NaCl tastant, whereas only 28.9% (± 11.3) of the cells in the 3D-Col I hydrogel responded to NaCl (Fig. 7G). Our results indicate an

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improvement of tastant sensing in the taste cells cultured in the 3D TEM hydrogel, which probably results from the biochemical and biophysical properties of the 3D TEM hydrogels

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mimicking the 3D microenvironments of the surrounding taste cells of the native tongue [23, 52]. In future studies, it would be worth investigating which factors in the 3D TEM hydrogel are critical for improving the viability, functionality, and sensitivity of taste cells.

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3.7. Microfluidic taste devices with taste cells and tongue-like microenvironments

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Finally, artificial tongue devices equipped with actual cellular and extracellular components for taste sensing in 3D tongue-specific microenvironments were established in microfluidic

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systems with primary taste cells and TEM. In view of industrial considerations, artificial taste sensors are needed to generate the objective and reproducible data required for the

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development and quality control of food products. Previously reported artificial sensors based on taste receptor-expressing cells have had limited success in reconstructing the cellular phenotypes, complex sensing mechanisms, and simultaneous responses of native taste cells [18]. These issues may limit the performance of currently available cell-based taste sensors for the precise, sensitive detection of specific tastants or other molecules and the generation of a reproducible data set [18]. Moreover, most cell-based sensor systems require bulky electrochemical detection devices [15, 16, 53]. Hence, we demonstrated here a functional tongue-mimicking microfluidic system that is highly efficient, is sensitive to tastant 25

ACCEPTED MANUSCRIPT molecules, and can analyze and interpret data without the need for additional detection devices (Fig. 8A). Microfluidic systems are advantageous to industry because they are costeffective and can be utilized for high-throughput screening using a small volume of test

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ingredients [26, 54]. In terms of cell and tissue engineering, microfluidic systems can provide versatile platforms for lifelike 3D models and organ-on-a-chip systems by enabling the reconstitution of tissue-specific microenvironments in the devices [26, 54-56].

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The microfluidic systems with primary taste cells and TEM markedly improved the taste-sensing performance of the reconstituted artificial tongue devices. To prepare

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microfluidic taste devices, TEM (0.02 and 0.1 mg/mL) or Col I (0.02 mg/mL) was coated onto the surfaces of the microchannels in the microfluidic devices (Fig. S4), which were constructed from PDMS, and the primary taste cells were cultured on the ECM-coated channel surfaces in the devices. After 3 days of culture, the expression levels of the cell-

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specific phenotypic markers gustducin and PLC-β2 were much greater in the TEM (0.1)

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group than in the Col I and TEM (0.02) groups (Fig. 8B). When the taste-sensing functionality of the microfluidic systems was evaluated by treatment with salty (NaCl) and

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sweet (sucrose) compounds, and the subsequent calcium influx imaging analysis was carried out, we observed a much higher level of Ca2+ influx intensity in the TEM (0.1) group than in

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the Col I and TEM (0.02) groups, regardless of the type of tastant (Fig. 8C, D, F, G). The percentage of tastant-responsive cells also increased in the TEM groups (Fig. 8E, H). Interestingly, a higher concentration of TEM (0.1 mg/mL) was found to be most effective for improving the phenotype and sensitivity of the taste cells in the microfluidic system, whereas a lower concentration of TEM (0.02 mg/mL) was more effective for taste sensing than a higher concentration (0.1 mg/mL) in the 2D bulk scenario. The discrepancy in the optimal TEM concentration between the culture conditions (2D bulk versus 3D microscale culture) should be further elucidated in future studies that compare 2D bulk and 3D microfluidic 26

ACCEPTED MANUSCRIPT systems for taste-sensing efficacy. The results from our microfluidic studies show that the phenotypic characteristics and the functionality of primary taste cells for taste sensing can be improved by incorporating tissue-derived matrix components in the microfluidic systems,

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implying the potential usefulness of microfluidic devices as high-throughput, standardized taste sensor platforms.

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4. Conclusions

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For the last few decades, artificial taste evaluation systems have been required for detecting and quantifying specific molecules in food, beverages, and pharmacological products. This has contributed to the development of new products and improved quality control. Although there have been thorough studies and trials to develop sensors and devices that are capable of

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mimicking the functions of the tongue, the difficulties presented by the in vitro reconstitution

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of complicated cellular signaling cascades for taste sensing have hindered the realization of highly efficient and sensitive taste sensor systems. Furthermore, the performance of cell-

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based taste sensor systems has been limited owing to the lack of techniques that facilitate the stable, long-term culture of functional taste cells that retain taste cell-specific phenotypes and

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properties. To overcome these limitations of current taste-sensing devices, we herein established functional 2D and 3D platforms based on decellularized tongue-derived matrix, which can provide tongue-like environmental control and thus facilitate primary taste cell culture and sensitive taste perception. The developed TEM-based platforms greatly improve cellular adhesion and maintain the functional phenotypes of primary taste cells, enabling the highly efficient, sensitive detection of various tastant compounds. We also demonstrated the potential usefulness of the TEM-based systems for industrial applications by investigating market products that require continuous quality control, i.e., coffee and wine. Interestingly, 27

ACCEPTED MANUSCRIPT we found that taste signal transduction from activated taste cells to neurons could be simulated in the TEM-based systems. Finally, microfluidic devices combined with TEM demonstrated great potential as economical taste-sensing units with high sensitivity. Our

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artificial tongue reconstructed with TEM and primary taste cells could provide highly efficient, sensitive taste devices, and efficient in vitro models for studying the mechanisms underlying the signal cascades and complex intracellular interactions in taste perception.

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Although here we aimed to check the potential of TEM for generating sensitive and multi-responsive primary taste cell-based artificial tongue system, investigation of signal

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pathways related to tongue development and taste organ homeostasis and application of taste stem or progenitor cells would broaden the utility of TEM-based systems to generate in vivolike tongue-mimicking taste platforms with maturity and functionality. Actually, it has been known that several signal pathways are critical for tongue development, tissue integrity, and

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proliferation and differentiation of taste bud cells or taste stem/progenitor cells. For example,

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Notch signaling has been known to be a crucial pathway in proliferation of Pax7-expressing muscle stem cells or progenitor cells and regulation of tongue development [57]. Wnt

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signaling pathway is also closely associated with lingual papillae development [57-59]. Hedgehog (Hh) signaling pathway is an essential regulator of cellular and functional integrity

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in taste system [60]. In this study, we did not assess regulation of these signaling pathways by TEM-based systems, but it would be valuable to investigate the correlation between TEM and improved phenotypic maintenance and functionality of taste cells in terms of various signaling pathways including Wnt/β-catenin, Hh, BMP, Notch, and FGF in future studies. Activation of these pathways such as Notch/Pax7, Hh, and canonical Wnt signaling in TEMbased systems could generate artificial taste systems with more mature and functional lingual taste papillae and further improve taste bud progenitor cell proliferation and differentiation, and physiological functions. TEM-based cell culture system would also be exploited with the 28

ACCEPTED MANUSCRIPT lingual organoids, which have recently been highlighted as a taste model system mimicking taste bud-like 3D structures and demonstrating regenerative capacity and physiological functions of tongue tissue [61]. Tongue-mimicking artificial taste models could be

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established with lingual taste bud organoids to elucidate the mechanisms of tongue development and provide in vivo-like taste organ models. These future studies would broaden the application of TEM to lingual organoid culture, taste tissue regeneration, and functional

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taste cell differentiation from stem cells or progenitor cells.

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Acknowledgements

J. S. Lee and A.-N. Cho made equal contributions to this work. The work was supported by the Samsung Research Funding Center of Samsung Electronics under Project Number SRFC-

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Fig. 1. Preparation and characterization of TEM-based platforms for taste cell culture. (A) Schematic illustration of taste cell- and TEM-based taste sensing systems. (B) Characterization of tongue tissue matrix before and after decellularization. Gross view (left panel), H&E staining (middle panel), and immunohistochemical staining for Col I (right

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panel). Scale bars = 400 μm. The quantification of (C) DNA and (D) GAG contents in native or decellularized tongue matrix (n = 3, * and ** indicate p-values < 0.05 and < 0.01,

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respectively, compared with the tissue before decellularization). (E) XPS analysis for comparing surface atomic composition of non-coated and TEM-coated (0.02 and 0.1 mg/mL)

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polystyrene cell culture substrates. (F) Measurement of water contact angle on each substrate (n = 6).

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Fig. 2. Enhanced adhesion and phenotypic characteristics of taste cells cultured on TEM-

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coated 2D platform. (A) Immunostaining for vinculin (green) and F-actin (red) of taste cells cultured on Col I, TEM (0.02), and TEM (0.1) surfaces 1 and 24 h after cell seeding. DAPI

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(blue) was used to counterstain nuclei. Scale bar = 100 μm. (B) Quantification of cell adhesion area in Col I, TEM (0.02), and TEM (0.1) groups at 1 and 24 h. The whole area of

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the images was considered to be 100% (n = 5, ** indicates a p-value < 0.01, compared with the Col I group at the same time-point). (C) Proliferation of taste cells in each group 1 and 3 days after cell seeding, which was measured using MTT assay (n = 4). (D) Immunostaining of taste cell-specific markers, gustducin (green) and PLC-β2 (red) after 3 days of culture. Scale bars = 100 μm. (E) qPCR analysis to quantify the gene expression of the taste cellspecific marker gustducin (GNAT3) in each group after 3 days of culture (n = 3, * and ** indicate p-values of < 0.05 and < 0.01, respectively, compared with the Col I group).

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Fig. 3. Evaluation of tastant sensing performance of taste cells on 2D tongue extracellular matrix (TEM)-based platform through imaging analysis of calcium influx changes. Activation of taste cells on Col I and TEM-coated substrates after treating with various tastants (NaCl for saltiness (A-C), sucrose for sweetness (D-F), saccharin for bitterness (G-I), and glycine for umami (J-L) taste) was visualized and quantified using calcium imaging analysis with a calcium indicator (Fluo-4). (A, D, G, J) Fluo-4-mediated visualization of cytosolic Ca2+ influx before and after (at peak intensity) tastant treatments. Scale bars = 100 39

ACCEPTED MANUSCRIPT μm. (B, E, H, K) The relative calcium influx intensity of the representative taste cells responding to tastant molecules (indicated by white arrows in Fig. 3A, D, G, J) is shown. (C, F, I, L) The percentage ratio of the responsive cells after tastant treatments (n = 4, * and **

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indicate p-values < 0.05 and < 0.01, respectively, compared with the Col I group).

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Fig. 4. Evaluation of taste cell responses to commercial products on TEM-based 2D platform. (A, D) Fluo-4-mediated visualization of cytosolic Ca2+ influx in taste cells on Col I- and

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TEM-coated substrates before and after (at peak intensity) tastant treatments (coffee and

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wine). Scale bars = 100 μm. (B, E) The relative calcium influx intensity of the representative responding taste cells (indicated by white arrows in Fig. 4A, D) is shown. (C, F) The

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percentage ratio of the responsive cells after tastant treatments (n = 4, ** indicates a p-value

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< 0.01, compared with the Col I group).

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Fig. 5. Reconstitution of taste signal transmission between taste cells and neurons on TEM-

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based 2D platform. The change in cytosolic Ca2+ concentration after treating with 5 μM

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denatonium benzoate was observed by Fluo-4-mediated calcium imaging analysis. (A) Gross view and fluorescence images of taste cells (labeled with DiI dye (red)) and neurons on TEM

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substrates. Scale bar = 100 μm. (B) Fluo-4-mediated visualization of cytosolic Ca2+ influx in taste cells and neurons after tastant treatment is presented. Scale bar = 100 μm. (C) The relative calcium influx intensity of the representative cells (taste cells responding to tastant molecules and neurons stimulated by activated taste cells) on TEM substrates (indicated by arrows in Fig. 5B). (D) Serial changes of cytosolic Ca2+ concentration along neurites (enlarged images of square box in Fig. 5B). Scale bar = 50 μm.

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Fig. 6. Characterization of TEM-based 3D hydrogel platform for taste cell culture. (A) SEM images and gross views (insets) of 3D-Col I, 3D-TEM (5), and 3D-TEM (10) hydrogel

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constructs. Scale bars = 1 μm (upper) and 200 nm (lower). (B) Rheological analysis of each hydrogel in frequency sweep mode (0.1–1 Hz). Elastic modulus (G′) and viscous modulus

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(G′′) were recorded for each hydrogel using a rheometer. (C) The average elastic modulus (G) of each hydrogel measured at 1 Hz frequency (n = 3, ** indicates p-value < 0.01, compared with the 3D-Col I and 3D-TEM (5) groups).

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Fig. 7. Enhanced viability, phenotypic maintenance, and taste sensing ability of taste cells in TEM-based 3D hydrogel platform. (A) Live/Dead staining of taste cells cultured in 3D-Col I

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and 3D-TEM (10) hydrogel at Days 0, 1, 3, and 5. Scale bar = 200 μm. (B) Quantification of cell viability in each hydrogel system (n = 4, * and ** indicate p-values < 0.05 and < 0.01, respectively, compared with the Col I group at the same time-point). (C) Immunostaining of taste cell-specific markers gustducin (green) and PLC-β2 (red) in taste cells in 3D hydrogels after 3 days of culture. Nuclei were counterstained with DAPI (blue). Scale bar = 200 μm. (D) Quantification of cell population expressing taste cell-specific markers in 3D-Col I and 3D-TEM (10) hydrogels at day 3 (n = 4, ** indicates p-value < 0.01, compared with the 3DCol I group). (E) Fluo-4-mediated visualization of cytosolic Ca2+ influx before and after (at 44

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tastant treatment (n = 4, ** indicates p-value < 0.01, compared with the 3D-Col I group).

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Fig. 8. Microfluidic artificial tongue device equipped with TEM and primary taste cells. (A)

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A scheme of microfluidic taste device with taste cells in tongue-like microenvironments. (B) Immunostaining of taste cell-specific markers gustducin (green) and PLC-β2 (red), in taste

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cells after 3 days of culture in the microfluidic device with TEM-deposited microchannels. DAPI (blue) was used to counterstain nuclei. Scale bar = 100 μm. (C, F) Fluo-4-mediated visualization of cytosolic Ca2+ influx before and after (at peak intensity) tastant treatments (NaCl and sucrose). Scale bars = 100 μm. (D, G) The relative calcium influx intensity of the representative taste cells responding to tastant molecules (indicated by white arrows in Fig. 8C, F). (E, H) The percentage ratios of the responsive cells after tastant treatment (n = 4, ** indicates p-value < 0.01, compared with the Col I group).

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