Name: Generation and Culture of Lingual Organoids Derived from Adult Mouse Taste Stem Cells (Video) | JoVE Journal
Uploaded: 2021-04-05T15:00:00.000+00:00
Duration: 7 min 57 s
Description: Learn to generate lingual organoids from adult mouse taste stem cells using FACS isolation and 3D culture with optimized WENRAS media. This protocol enables high-throughput screening of taste cell development and drug effects.

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5 minutes

The authors would like to thank Dr. Peter Dempsey and Monica Brown (University of Colorado Anschutz Medical Campus Organoid and Tissue Modeling Shared Resource) for providing WNR conditioned media and valuable discussions. We also thank the University of Colorado Cancer Center Cell Technologies and Flow Cytometry Shared Resources, especially Dmitry Baturin, for cell sorting expertise. This work was funded by: NIH/NIDCD R01 DC012383, DC012383-S1, DC012383-S2, and NIH/NCI R21 CA236480 to LAB, and R21DC016131 and R21DC016131-02S1 to DG, and F32 DC015958 to EJG.

All the animal procedures were performed in an AAALAC-accredited facility in compliance with the Guide for the Care and Use of Laboratory Animals, Animal Welfare Act, and Public Health Service Policy, and were approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Colorado Anschutz Medical Campus. Lgr5EGFP-IRES-CreERT2 mice used in this protocol are from The Jackson Laboratory, Stock No. 008875.
NOTE: The following steps should be completed before beginning to ensure smooth and timely progression of the protocol: set water bath to 37 &#176;C, set centrifuge to 4 &#176;C, make injection and dissociation enzyme solutions from the 10 mg/mL Dispase, Collagenase, and Elastase stock solutions (see Table of Materials), remove matrix gel from -20 &#176;C freezer (~750 &#181;L needed for a 48-well plate) and thaw by submerging the vial in ice for at least 3-4 h, pre-coat microcentrifuge tubes in undiluted FBS by rocking gently at room temperature for at least 30 min (two 2 mL tubes for tissue collection, two 1.5 mL tubes for dissociated cells, and one 1.5 mL tube for collection of single cells from cell sorter; remove excess FBS before use).
1. Isolation of CVP epithelium
NOTE: To obtain enough LGR5+ cells for a full 48-well plate, collect three Lgr5-EGFP CVPs in the same tube and process simultaneously. Importantly, harvest and process the CVP of at least one wild type littermate in parallel in a separate tube and utilize it as a gating control to set FACS parameters (see Representative Results).
<ol>
	<li>Euthanize the mice with CO2 asphyxiation according to IACUC regulations, followed by an approved secondary method such as bilateral thoracotomy, cervical dislocation, decapitation, or exsanguination.</li>
	<li>Use large sterile dissection scissors to cut the cheeks and break the jaw. Lift the tongue and cut the lingual frenulum to separate the tongue from the floor of the oral cavity. Cut out the tongue and collect it in sterile ice-cold Dulbecco&#39;s phosphate-buffered saline (dPBS) with Ca2+ and Mg2+.</li>
	<li>Remove and discard the anterior tongue by cutting just anterior of the intermolar eminence with a razor blade (Figure 1A, dashed line). Use a delicate task wipe to remove any hair and excess liquid from the posterior tongue.</li>
	<li>Fill 1 mL syringe with 200-300 &#181;L of injection enzyme solution (final concentration: 2 mg/mL type-I Collagenase and 5 mg/mL Dispase II in Ca2+/Mg2+-containing dPBS, diluted from 10 mg/mL stock solutions) and insert a 30 G x &#189; needle just above the intermolar eminence (Figure 1B, black arrow) until just anterior to&#160;the CVP (Figure 1B, black box). Inject the enzyme solution underneath and at the lateral edges of the CVP between the epithelium and the underlying tissues (lamina propria, muscle). Withdraw the syringe slowly and continuously from the tongue as the injection is performed.</li>
	<li>Incubate the tongue in sterile Ca2+/Mg2+-free dPBS at room temperature for precisely 33 min.</li>
	<li>Make small cuts in the epithelium bilaterally and just anterior to&#160;the CVP using extra fine dissection scissors, and gently peel the epithelium by lifting it with fine forceps. Once the trench epithelium is free of the underlying connective tissue, place it in an empty 2 mL microcentrifuge tube pre-coated with FBS. Do the epithelial trimming before or after detaching the CVP epithelium (Figure 1C,D).</li>
</ol>
2. Dissociation of CVP epithelium
NOTE: Dissociation of the CVP epithelium and plating are represented graphically in Figure 2.
<ol>
	<li>Add the dissociation enzyme cocktail (final concentration: 2 mg/mL type-I Collagenase, 2 mg/mL Elastase, and 5 mg/mL Dispase II in Ca2+/Mg2+-containing dPBS, diluted from 10 mg/mL stock solutions) to tubes containing peeled CVP epithelia (200 &#181;L per CVP). Incubate in a 37 &#176;C water bath for 45 min. Vortex briefly every 15 min. 
	NOTE: Prewarm 0.25% Trypsin-EDTA in 37 &#176;C water bath during the last 15 min of enzyme cocktail incubation.</li>
	<li>Following incubation, vortex (three pulses) then triturate with a glass Pasteur pipette for 1 min. After tissue pieces settle, pipette the supernatant containing first collection of dissociated cells, into new FBS-coated 1.5 mL microcentrifuge tubes corresponding to the genotype. Process the remaining tissue pieces further as described in step 2.3. below.
	<ol>
		<li>Spin the supernatant for 5 min at 370 x g and 4 &#176;C to pellet cells.</li>
		<li>Remove the resulting supernatant and resuspend the cell pellet in Fluorescence-Activated Cell Sorting (FACS) Buffer (1 mM EDTA, 25 mM HEPES (pH 7.0) and 1% FBS in Ca2+/Mg2+-free PBS (50 &#181;L per CVP)). Store on ice.</li>
	</ol>
	</li>
	<li>While carrying out steps 2.2.1 and 2.2.2, dissociate the remaining tissue pieces from step 2.2 by adding pre-warmed 0.25% trypsin-EDTA (200 &#181;L per CVP) to the original 2 mL microcentrifuge tubes and incubate in a 37 &#176;C water bath for 30 min. Vortex briefly every 10 min.</li>
	<li>Following incubation, vortex the tube containing tissue pieces (three pulses) then triturate with a glass Pasteur pipette for 1 min. After tissue pieces settle, pipette the supernatant into the 1.5 mL microcentrifuge tubes containing cells from step 2.2.2. Discard the tubes containing the remaining tissue pieces.
	<ol>
		<li>Spin the tubes with dissociated cells for 5 min at 370 x g and 4 &#176;C to pellet cells.</li>
		<li>Remove the supernatant and resuspend cell pellets in FACS Buffer (100 &#181;L per CVP). Store on ice.</li>
	</ol>
	</li>
	<li>Pass the cells through a 30 &#181;m nylon mesh filter and add DAPI (&#955;emission = 450 nm) to cell mixtures prior to FACS. Isolate Lgr5-GFP+ cells via FACS using the green fluorescent protein channel (&#955;excitation = 488 nm; &#955;emission = 530 nm). Sort the cells using a 100 &#181;m nozzle into a fresh FBS-coated 1.5 mL microcentrifuge tube containing 300 &#181;L of Ca2+/Mg2+ free dPBS. Place the cells on ice until plating.</li>
</ol>
3. Plating of Lgr5-EGFP cells
<ol>
	<li>Determine the volume of LGR5+ cell suspension received from the flow cytometer.</li>
	<li>Based on the number of cells obtained from the sorter, calculate the number of cells per &#181;L. Then, determine the volume needed to obtain the desired number of cells for plating (we use 200 cells per well of a 48-well plate) and transfer that total volume of suspended cells into a new microcentrifuge tube.</li>
	<li>Spin the tube for 5 min at 370 x g and 4 &#176;C to pellet cells (pellet may not be visible). Remove the supernatant and place the tube on ice.</li>
	<li>Gently resuspend the cell pellet in the appropriate amount of matrix gel (15 &#181;L per well for 48-well plates); pipette up and down gently to thoroughly distribute cells in matrix gel. Place 15 &#181;L of matrix gel/cell mixture in the center of each well. Keep the microcentrifuge tube on ice in a 50 mL conical tube during plating to prevent matrix gel from gelling. Continue to mix matrix gel/cell mixture throughout plating by pipetting up and down every three wells to ensure an even distribution of cells across wells.</li>
	<li>Place the plate in the incubator (37 &#176;C, 5% CO2, ~95% humidity) for 10 min to allow matrix gel gelling. Then, add 300 &#181;L of room temperature WENRAS + Y27632 media to each well and return the plate to the incubator.</li>
</ol>
4. Organoid maintenance
NOTE: Organoids are grown in conventional organoid media (WENR) comprising recombinant EGF and 50% conditioned media containing Wnt3a, Noggin, and R-spondin23. Drugs A8301 and SB202190 are added for the first 6 days of the culture period to optimize growth (WENRAS media) (Figure 5), then removed to promote differentiation (WENR media)20. Y27632 is added for the first 2 days of culture to promote survival. Media conditions relative to the culture timeline are presented in Figure 4.
<ol>
	<li>Two days after plating, remove WENRAS + Y27632 media from each well using a 1 mL pipette, ensuring no cross-contamination. Add 300 &#181;L of WENRAS media down the side of the well to not disrupt the matrix gel. Return the plate to the incubator.</li>
	<li>Change the media every 2 days, using the appropriate media for the culture stage (Figure 4). Maintain organoids until day 12, when the organoids are ready to harvest.</li>
</ol>
5. RNA processing
<ol>
	<li>Harvesting organoids for RNA
	<ol>
		<li>Place&#160;48-well plate on ice for 30 min to depolymerize the matrix gel.</li>
		<li>Using a 1 mL pipette, pull up the organoid media; then, as the media is returned to the well, use the tip of the pipette to scratch and further break up the matrix gel. Transfer the contents to a 1.5 mL microcentrifuge tube, pooling contents of three wells in one tube. Centrifuge the tubes for 5 min at 300 x g at room temperature.</li>
		<li>Remove as much media supernatant as possible without removing any organoids; then, spin down tubes again for 5 min at 300 x g at room temperature.</li>
		<li>Remove the remaining media and resuspend the organoids in 350 &#181;L lysis buffer + &#946;-mercaptoethanol (&#946;ME) (10 &#181;L &#946;ME per 1 mL lysis buffer). Place the samples on ice for immediate RNA extraction or store at -80 &#176;C.</li>
	</ol>
	</li>
	<li>Quantitative RT-PCR analysis
	<ol>
		<li>Measure RNA quantity via spectrophotometer. Reverse-transcribe RNA using a cDNA Synthesis Kit.</li>
		<li>Mix cDNA equivalent to 5 ng RNA with 200 nM pre-validated forward and reverse primers (Table 1) and fluorescent PCR Master Mix. Run the qRT-PCR reaction for 40 cycles at: 95 &#176;C for 15 s, then 60 &#176;C for 60 s.</li>
	</ol>
	</li>
</ol>
6. Immunohistochemistry
<ol>
	<li>Harvesting and fixing the organoids
	<ol>
		<li>Place 48-well plate on ice for 30 min to loosen the matrix gel.</li>
		<li>Remove the organoid media and add 400 &#181;L of ice-cold PBS to each well. Then, remove PBS and add 400 &#181;L of ice-cold Cell Recovery Solution to each well. Rock gently at 4 &#176;C for 30 min.</li>
		<li>Coat a 1 mL pipet tip with 1% BSA in PBS, and gently pipet contents of the well up and down to break up the matrix gel. Transfer the organoids to a 1.5 mL microcentrifuge tube placed on ice.</li>
		<li>Rinse each well with 300 &#181;L PBS + 1% BSA and transfer any remaining organoids to the corresponding tubes. Remove Cell Recovery Solution/PBS + BSA from each tube. Add 400 &#181;L of ice-cold PBS, then repeat with another ice-cold PBS rinse.</li>
		<li>Remove PBS and fix organoids with 300 &#181;L ice-cold 4% PFA (in 0.1 M PB) for 20 min, incubating at room temperature. Remove PFA and rinse organoids with 400 &#181;L ice-cold PBS.</li>
		<li>Remove PBS; then, add 400 &#181;L PBS + 1% BSA. Store at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Immunofluorescence staining
	<ol>
		<li>Rinse organoids in 500 &#181;L PBS + 1% BSA. Then, incubate organoids in blocking solution (5% normal goat or donkey serum, 1% bovine serum albumin, 0.3% Triton X 100 in 1x PBS pH 7.3) for 2 h, rocking gently at room temperature.</li>
		<li>Add the primary antibody solution (primary antibodies diluted in blocking solution) and rock gently for 3 nights at 4 &#176;C.</li>
		<li>Wash organoids 4x for 1 h with 500 &#181;L PBS + 0.2% Triton, rocking gently at room temperature. Add secondary antibody solution (secondary antibodies diluted in blocking solution) and rock organoids overnight, protected from light, at 4 &#176;C.</li>
		<li>Wash organoids 3x for 1 h with 500 &#181;L PBS + 0.2% Triton, protected from light and rocking gently at room temperature. Incubate with DAPI diluted 1:10,000 in 0.1 M PB for 20 min, rocking and covered at room temperature.</li>
		<li>Wash the organoids 3x for 20 min with 0.1 M PB, rocking gently and covered at room temperature.</li>
	</ol>
	</li>
	<li>Slide mounting of organoids for inverted confocal microscopy. 
	&#8203;NOTE: Step-by-step pictures of the slide mounting process are shown in Figure 7.
	<ol>
		<li>Create a ~1 mm thick 22 x 22 mm square perimeter of non-toxic modeling clay on a microscope slide.</li>
		<li>Remove 0.1 M PB from microcentrifuge tube, and gently resuspend organoids in 100 &#181;L mounting medium of choice; then, transfer to center of the clay square.</li>
		<li>Fill the clay square until the mounting medium is almost to the top. Then, place 22 x 22 mm square coverslip over clay and press down firmly on the sides of the coverslip to seal. Let it cure according to the manufacturer&#39;s instructions (here, room temperature for 1-2 days) and store at 4 &#176;C.</li>
	</ol>
	</li>
</ol>

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<li>Dekkers, J. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High-resolution+3D+imaging+of+fixed+and+cleared+organoids%5BTitle%5D)+AND+%22Nature+Protocols%22%5BJournal%5D)">High-resolution 3D imaging of fixed and cleared organoids.</a> Nature Protocols. 14 (6), 1756-1771 (2019).</li>
<li>Fujii, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Human+intestinal+organoids+maintain+self-renewal+capacity+and+cellular+diversity+in+niche-inspired+culture+condition%5BTitle%5D)+AND+%22Cell+Stem+Cell%22%5BJournal%5D)">Human intestinal organoids maintain self-renewal capacity and cellular diversity in niche-inspired culture condition.</a> Cell Stem Cell. 23 (6), 787-793 (2018).</li>
<li>Clevers, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Modeling+development+and+disease+with+organoids%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Modeling development and disease with organoids.</a> Cell. 165 (7), 1586-1597 (2016).</li>
<li>Castillo-Azofeifa, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sonic+hedgehog+from+both+nerves+and+epithelium+is+a+key+trophic+factor+for+taste+bud+maintenance%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">Sonic hedgehog from both nerves and epithelium is a key trophic factor for taste bud maintenance.</a> Development. 144 (17), Cambridge, England. 3054-3065 (2017).</li>
<li>Vintschgau, M. v, Hönigschmied, J. Nervus glossopharyngeus und schmeckbecher. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aVintschgau%2C+Mv+%22Archiv+f%C3%BCr+die+gesamte+Physiologie+des+Menschen+und+der+Tiere%22">Archiv für die gesamte Physiologie des Menschen und der Tiere</a>. 14, 443-448 (1877).</li>
<li>Liu, H. X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Multiple+Shh+signaling+centers+participate+in+fungiform+papilla+and+taste+bud+formation+and+maintenance%5BTitle%5D)+AND+%22Developmental+Biology%22%5BJournal%5D)">Multiple Shh signaling centers participate in fungiform papilla and taste bud formation and maintenance.</a> Developmental Biology. 382 (1), 82-97 (2013).</li>
<li>Workman, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Engineered+human+pluripotent-stem-cell-derived+intestinal+tissues+with+a+functional+enteric+nervous+system%5BTitle%5D)+AND+%22Nature+Medicine%22%5BJournal%5D)">Engineered human pluripotent-stem-cell-derived intestinal tissues with a functional enteric nervous system.</a> Nature Medicine. 23 (1), 49-59 (2017).</li>
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</ol>

The authors have nothing to disclose.

Reported here is an efficient and readily repeatable method for culturing, maintaining, and processing lingual organoids derived from adult mouse taste stem cells. It was found that using three CVPs from 8 to 20-week-old Lgr5-EGFP mice is sufficient to obtain ~10,000 GFP+ cells for experimental use, resulting in 50 wells plated at a density of 200 cells per well in 48-well plates. Removal of CVP trench epithelia is optimized by injecting the lingual epithelium with freshly made Dispase II and type-I C...

In rodents, lingual taste buds are housed in fungiform papillae distributed anteriorly, bilateral foliate papillae posteriorly, as well as a single circumvallate papilla (CVP) at the posterodorsal midline of the tongue1. Each taste bud is composed of 50-100 short-lived, rapidly renewing taste receptor cells (TRCs), which include type I glial-like support cells, type II cells that detect sweet, bitter, and umami, and type III cells that detect sour2,3,4. In the mouse CVP, LGR5+ stem cells along the basal lamina produce all TRC types as well as non-taste epithelial cells5. When renewing the taste lineage, LGR5 daughter cells are first specified as post-mitotic taste precursor cells (type IV cells) that enter a taste bud and are capable of differentiating into any of the three TRC types6. The rapid turnover of TRCs renders the taste system susceptible to disruption by medical treatments, including radiation and certain drug therapies7,8,9,10,11,12,13. Thus, studying the taste system in the context of taste stem cell regulation and TRC differentiation is vital for understanding how to mitigate or prevent taste dysfunction.
Mice are a traditional model for in vivo studies in taste science since they have a taste system organized similarly to humans14,15,16. However, mice are not ideal for high throughput studies, as they are expensive to maintain and time-consuming to work with. To overcome this, in vitro organoid culture methods have been developed in recent years. Taste organoids can be generated from native CVP tissue, a process in which organoids bud off from isolated mouse CVP epithelium cultured ex vivo17. These organoids display a multilayered epithelium consistent with the in vivo taste system. A more efficient way to generate organoids that does not require ex vivo CVP culture was developed by Ren et al. in 201418. Adapting methods and culture media first developed to grow intestinal organoids, they isolated single Lgr5-GFP+ progenitor cells from mouse CVP and plated them in matrix gel19. These single cells generated lingual organoids that proliferate during the first 6 days of culture, begin to differentiate around day 8, and by the end of the culture period contain non-taste epithelial cells and all three TRC types18,20. To date, multiple studies utilizing the lingual organoid model system have been published17,18,20,21,22; however, methods and culture conditions used to generate these organoids vary across publications (Supplementary Table 1). Thus, these methods have been adjusted and optimized here to present a detailed standardized protocol for the culture of lingual organoids derived from LGR5+ progenitors of adult mouse CVP.
Lingual organoids provide a unique model for studying the cell biological processes driving taste cell development and renewal. As the applications of lingual organoids expand and more labs move toward utilizing in vitro organoid models, it is important that the field strives to develop and adopt standardized protocols to improve reproducibility. Establishing lingual organoids as a standard tool within taste science would enable high throughput studies that tease apart how single stem cells generate the differentiated cells of the adult taste system. Additionally, lingual organoids could be employed to rapidly screen drugs for potential impacts on taste homeostasis, which could then be investigated more thoroughly in animal models. This approach ultimately will enhance efforts to devise therapies that improve the quality of life for&#160;future drug recipients.

<table><tbody><tr><td>&lt;strong&gt;Antibodies&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Alexa Fluor 546 Donkey anti Goat IgG</td><td>Molecular Probes</td><td>A11056, RRID: AB_142628</td><td>1:2000</td></tr><tr><td>Alexa Fluor 546 Goat anti Rabbit IgG</td><td>Molecular Probes</td><td>A11010, RRID:AB_2534077</td><td>1:2000</td></tr><tr><td>Alexa Fluor 568 Goat anti Guinea pig IgG</td><td>Invitrogen</td><td>A11075, RRID:AB_2534119</td><td>1:2000</td></tr><tr><td>Alexa Fluor 647 Donkey anti Rabbit IgG</td><td>Molecular Probes</td><td>A31573, RRID:AB_2536183</td><td>1:2000</td></tr><tr><td>Alexa Fluor 647 Goat anti Rat IgG</td><td>Molecular Probes</td><td>A21247, RRID:AB_141778</td><td>1:2000</td></tr><tr><td>DAPI (for FACS)</td><td>Thermo Fischer</td><td>62247</td><td></td></tr><tr><td>DAPI (for immunohistochemistry)</td><td>Invitrogen</td><td>D3571, RRID:AB_2307445</td><td>1:10000</td></tr><tr><td>Goat anti-CAR4</td><td>R&amp;amp;D Systems</td><td>AF2414, RRID:AB_2070332</td><td>1:50</td></tr><tr><td>Guinea pig anti-KRT13</td><td>Acris Antibodies</td><td>BP5076, RRID:AB_979608</td><td>1:250</td></tr><tr><td>Rabbit anti-GUSTDUCIN</td><td>Santa Cruz Biotechnology Inc.</td><td>sc-395, RRID:AB_673678</td><td>1:250</td></tr><tr><td>Rabbit anti-NTPDASE2</td><td>CHUQ</td><td>mN2-36LI6, RRID:AB_2800455</td><td>1:300</td></tr><tr><td>Rat anti-KRT8</td><td>DSHB</td><td>TROMA-IS, RRID: AB_531826</td><td>1:100</td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>2D rocker</td><td>Benchmark Scientific Inc.</td><td>BR2000</td><td></td></tr><tr><td>3D Rotator</td><td>Lab-Line Instruments</td><td>4630</td><td></td></tr><tr><td>Big-Digit Timer/Stopwatch</td><td>Fisher Scientific</td><td>S407992</td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>5415D</td><td></td></tr><tr><td>CO2 tank</td><td>Airgas</td><td>CD USP50</td><td></td></tr><tr><td>FormaTM Series 3 Water Jackeed CO2 Incubator</td><td>Thermo Scientific</td><td>4110</td><td>184 L, Polished Stainless Steel</td></tr><tr><td>Incucyte</td><td>Sartorius</td><td>Model: S3</td><td>Cancer Center Cell Technologies Shared Resource, University of Colorado Anschutz Medical Campus</td></tr><tr><td>MoFlo XDP100</td><td>Cytomation Inc</td><td>Model: S13211997</td><td>&amp;nbsp;Gates Center Flow Cytometry Core, University of Colorado Anschutz Medical Campus</td></tr><tr><td>Orbital Shaker</td><td>New Brunswick Scientific</td><td>Excella E1</td><td></td></tr><tr><td>Real-Time PCR System</td><td>Applied Biosystems</td><td>4376600</td><td></td></tr><tr><td>Refrigerated Centrifuge</td><td>Eppendorf</td><td>5417R</td><td></td></tr><tr><td>Spectrophotometer</td><td>Thermo Scientific</td><td>ND-1000</td><td></td></tr><tr><td>&amp;nbsp;Stereomicroscope</td><td>Zeiss</td><td>Stemi SV6</td><td></td></tr><tr><td>Thermal Cycler</td><td>Bio-Rad</td><td>580BR</td><td></td></tr><tr><td>Vortex</td><td>Fisher Scientific</td><td>12-812</td><td></td></tr><tr><td>Water bath</td><td>Precision</td><td>51220073</td><td></td></tr><tr><td>&lt;strong&gt;Media&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>A83 01</td><td>Sigma</td><td>SML0788-5MG</td><td>Stock concentration 10 mM, final concentration 500 nM</td></tr><tr><td>Advanced DMEM/F12</td><td>Gibco</td><td>12634-010</td><td></td></tr><tr><td>B27 Supplement</td><td>Gibco</td><td>17504044</td><td>Stock concentration 50X, final concentration 1X</td></tr><tr><td>Gentamicin</td><td>Gibco</td><td>15750-060</td><td>Stock concentration 1000X, final concentration 1X</td></tr><tr><td>Glutamax</td><td>Gibco</td><td>35050061</td><td>Stock concentration 100X, final concentration 1X</td></tr><tr><td>HEPES</td><td>Gibco</td><td>15630080</td><td>Stock concentration 100X, final concentration 1X</td></tr><tr><td>Murine EGF</td><td>Peprotech</td><td>315-09-1MG</td><td>Stock concentration 500 &amp;micro;g/mL, final concentration 50 ng/mL</td></tr><tr><td>Murine Noggin</td><td>Peprotech</td><td>250-38</td><td>Stock concentration 50 &amp;micro;g/mL, final concentration 25 ng/mL</td></tr><tr><td>N-acetyl-L-cysteine</td><td>Sigma</td><td>A9165</td><td>Stock concentration 0.5 M, final concentration 1 mM</td></tr><tr><td>Nicotinamide</td><td>Sigma</td><td>N0636-100g</td><td>Stock concentration 1 M, final concentration 1 mM</td></tr><tr><td>Pen/Strep</td><td>Gibco</td><td>15140-122</td><td>Stock concentration 100X, final concentration 1X</td></tr><tr><td>Primocin</td><td>InvivoGen</td><td>ant-pm-1</td><td>Stock concentration 500X, final concentration 1X</td></tr><tr><td>SB202190</td><td>R&amp;amp;D Systems</td><td>1264</td><td>Stock concentration 10 mM, final concentration 0.4 &amp;micro;M</td></tr><tr><td>WRN Conditioned media</td><td></td><td></td><td>Received from Dempsey Lab (AMC Organoid and Tissue Modeling Share Resource). Derived from L-WRN (ATCC&amp;reg; CRL-3276&amp;trade;) cells</td></tr><tr><td>Y27632 dihydochloride 10ug</td><td>APExBIO</td><td>A3008-10</td><td>Stock concentration 10 mM, final concentration 10 &amp;micro;M</td></tr><tr><td>&lt;strong&gt;Other&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>1 ml TB Syringe</td><td>BD Syringe</td><td>309659</td><td></td></tr><tr><td>2-Mercaptoethanol, min. 98%</td><td>Sigma</td><td>M3148-25ML</td><td>&amp;beta;-mercaptoethanol</td></tr><tr><td>2.0 mL Microcentrifuge Tubes</td><td>USA Scientific</td><td>1420-2700</td><td></td></tr><tr><td>48-well plates</td><td>Thermo Scientific</td><td>150687</td><td></td></tr><tr><td>5 3/4 inch Pasteur Pipets</td><td>Fisherbrand</td><td>12-678-8A</td><td></td></tr><tr><td>Albumin from bovine serum (BSA)</td><td>Sigma Life Science</td><td>A9647-100G</td><td></td></tr><tr><td>Buffer RLT Lysis buffer</td><td>QIAGEN</td><td>1015750</td><td></td></tr><tr><td>Cell Recovery Solution</td><td>Corning</td><td>354253</td><td></td></tr><tr><td>Cohan-Vannas Spring Scissors</td><td>Fine Science Tools</td><td>15000-02</td><td></td></tr><tr><td>Collagenase from Clostridium histolyticum, type I</td><td>Sigma Life Science</td><td>C0130-1G</td><td></td></tr><tr><td>Cultrex RGF BME, Type 2, Pathclear</td><td>R&amp;amp;D Systems</td><td>3533-005-02</td><td>Matrigel</td></tr><tr><td>Dispase II (neutral protease, grade II)</td><td>Sigma-Aldrich (Roche)</td><td>4942078001</td><td></td></tr><tr><td>Disposable Filters</td><td>Sysmex</td><td>04-0042-2316</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s Phosphate Buffered Saline pH 7.4 (1X) (Ca&lt;sup&gt;2+&lt;/sup&gt; &amp;amp; Mg&lt;sup&gt;2+&lt;/sup&gt; free)</td><td>Gibco</td><td>10010-023</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s Phosphate Buffered Saline with Ca&lt;sup&gt;2+&lt;/sup&gt; &amp;amp; Mg&lt;sup&gt;2+&lt;/sup&gt;&amp;nbsp;</td><td>Sigma Life Sciences</td><td>D8662-500ML</td><td></td></tr><tr><td>Dumont #5 Forceps</td><td>Fine Science Tools</td><td>11252-30</td><td></td></tr><tr><td>EDTA, 0.5M (pH 8.0)</td><td>Promega</td><td>V4231</td><td></td></tr><tr><td>Elastase Lyophilized</td><td>Worthington Biochemical</td><td>LS002292</td><td></td></tr><tr><td>Extra Fine Bonn Scissors</td><td>Fine Science Tools</td><td>14084-08</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Gibco</td><td>26140-079</td><td></td></tr><tr><td>Fluoromount G</td><td>SouthernBiotech</td><td>0100-01</td><td></td></tr><tr><td>HEPES Solution</td><td>Sigma Life Science</td><td>H3537-100ML</td><td></td></tr><tr><td>HyClone Tryspin 0.25% + EDTA</td><td>Thermo Scientific</td><td>25200-056</td><td></td></tr><tr><td>iScript cDNA Synthesis Kit</td><td>Bio-Rad</td><td>1706691</td><td></td></tr><tr><td>Modeling Clay, Gray</td><td>Sargent Art</td><td>22-4084</td><td></td></tr><tr><td>Needle</td><td>BD Syringe</td><td>305106</td><td></td></tr><tr><td>Normal Donkey Serum</td><td>Jackson ImmunoResearch</td><td>017-000-121</td><td></td></tr><tr><td>Normal Goat Serum</td><td>Jackson ImmunoResearch</td><td>005-000-121</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma-Aldrich</td><td>158127</td><td></td></tr><tr><td>&lt;em&gt;Power&lt;/em&gt;SYBR Green PCR Master Mix</td><td>Applied Biosystems</td><td>4367659</td><td></td></tr><tr><td>RNeasy Micro Kit</td><td>QIAGEN</td><td>74004</td><td></td></tr><tr><td>Safe-Lock Tubes 1.5 mL</td><td>Eppendorf</td><td>022363204</td><td></td></tr><tr><td>Sodium Chloride</td><td>Fisher Chemical</td><td>7647-14-5</td><td></td></tr><tr><td>Sodium Phosphate dibasic anhydrous</td><td>Fisher Chemical</td><td>7558-79-4</td><td></td></tr><tr><td>Sodium Phosphate monobasic anhydrous</td><td>Fisher Bioreagents</td><td>7558-80-7</td><td></td></tr><tr><td>SuperFrost Plus Microscope Slides</td><td>Fisher Scientific</td><td>12-550-15</td><td></td></tr><tr><td>Surgical Scissors - Sharp</td><td>Fine Science Tools</td><td>14002-14</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma Life Science</td><td>T8787-100ML</td><td></td></tr><tr><td>VWR micro cover glass</td><td>VWR</td><td>48366067</td><td>22x22mm</td></tr></tbody></table>

generation and culture of lingual organoids derived from adult mouse taste stem cells

Generation and Culture of Lingual Organoids Derived from Adult Mouse Taste Stem Cells

The sense of taste is mediated by taste buds on the tongue, which are composed of rapidly renewing taste receptor cells (TRCs). This continual turnover is powered by local progenitor cells and renders taste function prone to disruption by a multitude of medical treatments, which in turn severely impacts the quality of life. Thus, studying this process in the context of drug treatment is vital to understanding if and how taste progenitor function and TRC production are affected. Given the ethical concerns and limited availability of human taste tissue, mouse models, which have a taste system similar to humans, are commonly used. Compared to in vivo methods, which are time-consuming, expensive, and not amenable to high throughput studies, murine lingual organoids can enable experiments to be run rapidly with many replicates and fewer mice. Here, previously published protocols have been adapted and a standardized method for generating taste organoids from taste progenitor cells isolated from the circumvallate papilla (CVP) of adult mice is presented. Taste progenitor cells in the CVP express LGR5 and can be isolated via EGFP fluorescence-activated cell sorting (FACS) from mice carrying an Lgr5EGFP-IRES-CreERT2 allele. Sorted cells are plated onto a matrix gel-based 3D culture system and cultured for 12 days. Organoids expand for the first 6 days of the culture period via proliferation and then enter a differentiation phase, during which they generate all three taste cell types along with non-taste epithelial cells. Organoids can be harvested upon maturation at day 12 or at any time during the growth process for RNA expression and immunohistochemical analysis. Standardizing culture methods for production of lingual organoids from adult stem cells will improve reproducibility and advance lingual organoids as a powerful drug screening tool in the fight to help patients experiencing taste dysfunction.

Mice have one CVP, located posteriorly on the tongue, from which LGR5+ stem cells can be isolated (Figure 1A, black box). Injection of an enzyme solution under and around the CVP (Figure 1B) results in slight swelling of the epithelium and digestion of the connective tissue. Sufficient digestion is achieved following a 33 min incubation, which allows easy separation of the CVP epithelium from the underlying tissue. When attempting to ...

The protocol presents a method for culturing and processing lingual organoids derived from taste stem cells isolated from the posterior taste papilla of adult mice.

The sense of taste is mediated by taste buds on the tongue, which are composed of rapidly renewing taste receptor cells (TRCs). This continual turnover is ...

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Research

JoVE Journal

Developmental Biology

We aimed to develop a reproducible protocol for isolating and maintaining long-term cultures of human fungiform taste papillae cells. Cells from human fungiform papillae obtained by biopsy were successfully maintained in culture for more than eight passages (12 months) without loss of viability.

Isolation and Culture of Human Fungiform Taste Papillae Cells

In this video, we describe the isolation of epithelial cells from a murine tongue. These cells can be further cultured to give rise to cell colonies.

Isolation and Culture of Murine Tongue Epithelial Cells: A Procedure to Isolate and Culture Tongue Epithelial Cells from Mouse Model

The continuously growing mouse incisor provides a model for studying renewal of dental tissues from dental epithelial stem cells (DESCs). A robust system for consistently and reliably obtaining these cells from the incisor and expanding them in vitro is reported here.

Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor

Stem cell derived cultures harbor tremendous potential to model infectious diseases. Here, the culture of mouse and human gastric organoids derived from adult stem cells is described. The organoids are microinjected with the gastric pathogen Helicobacter pylori.

Organoids as Model for Infectious Diseases: Culture of Human and Murine Stomach Organoids and Microinjection of Helicobacter Pylori

This paper describes methods for tissue preparation, staining, and analysis of whole fungiform, circumvallate, and palate taste buds that consistently yield whole and intact taste buds (including the nerve fibers that innervate them) and maintain the relationships between structures within taste buds and the surrounding papilla.

Whole-Mount Staining, Visualization, and Analysis of Fungiform, Circumvallate, and Palate Taste Buds

We have developed a generalized protocol to dissociate a large quantity of high-quality single cells from the epithelium and mesenchyme/connective tissue of embryonic and adult mouse tongues.

Cell Dissociation from the Tongue Epithelium and Mesenchyme/Connective Tissue of Embryonic-Day 12.5 and 8-Week-Old Mice

The Trowell-type organ culture method has been used to unravel complex signaling networks that govern tooth development and, more recently, for studying regulation involved in stem cells of the continuously growing mouse incisor. Fluorescent-reporter animal models and live-imaging methods facilitate in-depth analyses of dental stem cells and their specific niche microenvironment.

Use of Trowell-Type Organ Culture to Study Regulation of Dental Stem Cells

The present protocol describes the isolation and culture of oral keratinocytes derived from the adult mouse palate. An evaluation method using immunostaining is also reported.

Isolation and Culture of Primary Oral Keratinocytes from the Adult Mouse Palate

This protocol describes the key steps to generate and characterize murine oral-esophageal 3D organoids that represent normal, preneoplastic, and squamous cell carcinoma lesions induced via chemical carcinogenesis.

Modeling Oral-Esophageal Squamous Cell Carcinoma in 3D Organoids

Here we provide a reproducible method to examine adult neurogenesis using a neurosphere assay derived from the whole brain or from either the telencephalic, tectal or cerebellar regions of the adult zebrafish brain. Additionally, we describe the procedure to manipulate gene expression in zebrafish neurospheres.

Isolation and Culture of Adult Zebrafish Brain-derived Neurospheres

Learn to generate lingual organoids from adult mouse taste stem cells using FACS isolation and 3D culture with optimized WENRAS media. This protocol enables high-throughput screening of taste cell development and drug effects.

Generation and Culture of Lingual Organoids Derived from Adult Mouse Taste Stem Cells

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