Name: generation and culture of lingual organoids derived from adult mouse taste stem cells
Uploaded: 2021-04-05T15:00:00
Description: Watch this Scientific Journal Video about Generation and Culture of Lingual Organoids Derived from Adult Mouse Taste Stem Cells at JoVE.com

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.
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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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Antibodies</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&#38;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>Equipment</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>&#160;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>&#160;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>Media</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 &#181;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 &#181;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&#38;D Systems</td>
			<td>1264</td>
			<td>Stock concentration 10 mM, final concentration 0.4 &#181;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&#174; CRL-3276&#8482;) cells</td>
		</tr>
		<tr>
			<td>Y27632 dihydochloride 10ug</td>
			<td>APExBIO</td>
			<td>A3008-10</td>
			<td>Stock concentration 10 mM, final concentration 10 &#181;M</td>
		</tr>
		<tr>
			<td>Other</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>&#946;-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&#38;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&#8217;s Phosphate Buffered Saline pH 7.4 (1X) (Ca2+ &#38; Mg2+ free)</td>
			<td>Gibco</td>
			<td>10010-023</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Phosphate Buffered Saline with Ca2+ &#38; Mg2+&#160;</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>PowerSYBR 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

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 ...

Watch this Scientific Journal Video about Generation and Culture of Lingual Organoids Derived from Adult Mouse Taste Stem Cells at JoVE.com

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

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 ...

Organoids are powerful in vitro technology used for drug screening and understanding biological processes. So, generating organoids that model the tongue is crucial for studying taste cell development and regeneration. Traditional in vivo taste studies can be expensive and time-consuming.This organoid protocol offers a standardized, reproducible alternative that minimizes those challenges while allowing for higher throughput experiments. After euthanizing an adult mouse, use large sterile dissection scissors to cut the cheeks and break the jaw. Then, lift the tongue and cut the lingual frenulum to separate the tongue from the floor of the oral cavity.Cut the tongue out and collect it in sterile ice-cold dPBS with calcium and magnesium. Under a dissecting microscope, remove and discard the anterior tongue by cutting just anterior of the inter malar eminence with a razor blade. Then using a delicate task wipe to remove any hair and excess liquid from the posterior tongue.Next, fill a one milliliter syringe with 200 to 300 microliters of injection enzyme solution and insert a 30 gauge half-inch needle just above the inter malar eminence until just anterior of the circumvallate papillae, or CVP. Inject the enzyme solution underneath and at the lateral edges of the CVP between the epithelium and the underlying tissues. Withdraw the syringe slowly and continuously from the tongue as the solution is being injected.Incubate the tongue and sterile calcium magnesium-free dPBS at room temperature for precisely 33 minutes. Using extra-fine dissection scissors, make small cuts in the epithelium bilaterally and just anterior of the CVP. Then gently peel the epithelium by lifting it with fine forceps.Once the trench epithelium is free of the underlying connective tissue, place it into two milliliter microcentrifuge tube, pre-coated with FBS. Add the dissociation enzyme cocktail to the tubes containing the peeled CVP epithelia and incubate them in a 37 degree Celsius water bath for 45 minutes with brief vortexing every 15 minutes. During the last 15 minutes of incubation, prewarm 0.25%Trypsin-EDTA in the water bath.Following incubation, vortex the tubes in triturate with a glass Pasteur pipette for one minute. After tissue pieces settle, pipette the supernatant containing the first collection of dissociated cells into new FBS coated 1.5 milliliter microcentrifuge tubes. Spin the supernatant for five minutes at 370 times G and four degrees Celsius to pellet the cells.Remove the resulting supernatant, then resuspend the cell pellet in FACS buffer and keep it on ice. To dissociate the remaining tissue pieces in the original two milliliter microcentrifuge tubes, add pre-warmed 0.25%Trypsin-EDTA and incubate at 37 degrees Celsius for 30 minutes with brief vortexing every 10 minutes. Then, triturate the tissue pieces with a glass Pasteur pipette for one minute.After the tissue pieces settle, pipette the supernatant into the 1.5 milliliter microcentrifuge tubes containing the previously collected dissociated cells in FACS buffer. Spin the tubes with the dissociated cells. After removing the supernatant, resuspend the cell pellets in FACS buffer and keep them on ice.Then, isolate the Lgr5-GFP positive cells via FACS using the green fluorescent protein channel as described in the text manuscript. Transfer the desired number of Lgr5 positive suspended cells into a new microcentrifuge tube. Spin the tube for five minutes at 370 times G and four degrees Celsius to pellet the cells.Remove the supernatant and place the tube on ice. Gently resuspend the cell pellet in the appropriate amount of matrix gel with gentle pipetting. Then keep the microcentrifuge tube on ice in a 50 milliliter conical tube to prevent the matrix gel from gelling.Add 15 microliters of the matrix gel in cell mixture in the center of each well of a 48-well plate. To ensure an even distribution of cells, mix the matrix gel and cell mixture by pipetting up and down after plating every three wells. Place the plate in an incubator at 37 degrees Celsius, 5%carbon dioxide and 95%humidity for 10 minutes to allow gelling of the matrix gel.Then, add 300 microliters of room temperature WENRAS media supplemented with the rock inhibitor, Y27632, to each well and return the plate to the incubator. Two days after plating, remove the media from each well using a one milliliter pipette or via vacuum aspiration, ensuring no cross-contamination between conditions. Add 300 microliters of WENRAS media down the side of the well, taking care not to disrupt the matrix gel and return the plate to the incubator.When lingual organoids are cultured using WENR media, they do not grow efficiently. However, after adding A 83-01, a TGF-beta signaling inhibitor, and SB202190, P-38 map kinase signaling inhibitor, robust growth is observed. Interestingly, removing these inhibitors from the media after six days results in higher expression of general taste receptor cell marker, Kcnq1, suggesting that A 83-01 and SB-202190 hinder taste cell differentiation.Thus, optimal growth and differentiation are obtained by culturing organoids in WENRAS media from day zero to six and WENR media from day six to 12. Mature organoids contain both taste cells marked by keratin-8 and non-taste epithelial cells marked by keratin-13. Further, keratin-13 is expressed at higher levels than all three taste receptor cell markers, suggesting that organoids are predominantly composed of non-taste epithelial cells.The organoids express all taste receptor cell types. Type one cells marked by Entpd2 and bitter type two cells marked by Gnat3 are highly expressed in taste organoids, while sour sensing type three cells marked by Car4 are less common. The taste receptor cells are randomly distributed in the organoids rather than in discrete taste bud structures observed in vivo.Make sure to include some tissue posterior to the CVP when dissecting the tongue from the oral cavity. Additionally, make sure both trenches pop out and are obtained when peeling the epithelium.

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