This study was supported by the National Institutes of Health, grant number R01DC012308 and R21DC018089 to HXL. We give thanks to Brett Marshall (University of Georgia, Athens, GA) and Egon Ranghini (10X GENOMICS, Pleasanton, CA) for technical assistance and consultation regarding the cell dissociation; to Francisca Gibson Burnley (University of Georgia, Athens, GA) for English editing.

Animal use (C57BL/6 mice throughout the study) was approved by the University of Georgia Institutional Animal Care and Use Committee and was in accordance with the National Institutes of Health Guidelines for care and use of animals for research.
1. Animal usage
NOTE: Mice were bred and maintained in the animal facility of the Animal and Dairy Science department at the University of Georgia at 22 &#176;C under 12-h day/night cycles.
<ol>
	<li>Designate noon of the day of vaginal plug detection in mice as embryonic (E) day 0.5. Use embryos at E12.5 and postnatal mice at 8 weeks of age for the following experiments.</li>
</ol>
2. Preparation before experiment
NOTE: The instruments required for this protocol are listed in the Table of Materials.
<ol>
	<li>Autoclave instruments before the experiment. Sterilize instruments using a bead sterilizer during the experiment.</li>
	<li>Clean the surgical area, the dissecting microscope, and the biosafety cabinet using 70% ethanol&#160;wipes. Turn on the UV light of biosafety cabinet and keep it on for 20 min prior to the experimental procedure.</li>
	<li>Prepare an enzyme mixture of 1:1 dispase (5.0 mg/mL) and collagenase (2.0 mg/mL) to a final concentration of 2.5 and 1.0 mg/mL respectively, and filter the solution using 0.22 &#181;m syringe filter7.
	<ol>
		<li>Prepare 1 mL of enzyme mixture for an adult tongue or 0.5 mL for a specific region of tongue (e.g., posterior, or anterior tongue).</li>
		<li>Prepare 2 mL of enzyme mixture for embryonic tongues.</li>
	</ol>
	</li>
	<li>Make 10 mL of 2.5% BSA in 0.1 M PBS and filter the solution using 1 mL syringe and 0.22 &#181;m syringe filter.</li>
	<li>Make 500 &#181;L of 5% FBS in DMEM/F12.</li>
	<li>Make 3 mL of DMEM/F12 containing 10% FBS and 1% BSA and filter the solution using 1 mL syringe and 0.22 &#181;m syringe filter.</li>
</ol>
3. Separation of the tongue epithelium from the mesenchyme/underlying connective tissue
<ol>
	<li>Separation of the epithelium from the mesenchyme of an E12.5 mouse tongue
	<ol>
		<li>Euthanize timed pregnant female mice carrying E12.5 embryos by placing it in a CO2 chamber followed by cervical dislocation. 
		NOTE: Mouse embryos at E12.5 are collected after 12 pm (afternoon) on the 12th day after detection of vaginal plug in the pregnant female mice.</li>
		<li>Transfer mice to the surgical area. Wet the mouse abdomen using 70% ethanol to prevent fur from getting into the operating site.</li>
		<li>Open the abdomen using dissecting scissors to expose the uterine horns carrying embryos. Dissect the uterine horns using dissecting scissors and transfer it to 15 mL of fresh Tyrode&#39;s solution in a 100 mm culture dish.</li>
		<li>Dissect the embryos (Figure 1A1) out from uterine horns under a dissecting microscope using mini-scissors and fine forceps.</li>
		<li>Carefully open the mouth cavity wide by using fine forceps and dissect the tongue off from the mandible using mini-scissors (Figure 1A2).</li>
		<li>Wash the tongues using 15 mL of fresh sterile Tyrode&#39;s solution in a 100 mm culture dish.</li>
		<li>Transfer the tissues to 2 mL of enzyme mixture of dispase (2.5 mg/mL) and collagenase (1.0 mg/mL) in a 35 mm culture dish with a spatula and fine forceps in a biosafety cabinet. Incubate for&#160;20 min at 37 &#176;C.</li>
		<li>Transfer tongues to 15 mL of fresh sterile Tyrode&#39;s solution in a 100 mm culture dish and gently remove the mesenchyme from the epithelium from the ventral side using fine forceps. 
		&#8203;NOTE: Epithelial sheets may be separated without mechanical force during the incubation.</li>
		<li>Wash the separated epithelia and mesenchyme twice in 15 mL of fresh sterile Tyrode&#39;s solution in a 100 mm culture dish. 
		NOTE: The activities of dispase and collagenase will be inhibited by EDTA in the cell dissociation procedure (step 4.1).</li>
	</ol>
	</li>
	<li>Separation of the tongue epithelium from the underlying connective tissue of adult mice
	<ol>
		<li>Euthanize the mouse at 8 weeks of age by placing it in a CO2 chamber. Confirm that the mouse is euthanized with no breaths and forepaw-pinch response.</li>
		<li>Transfer the mice to the surgical area. Wet the mouse head using 70% ethanol to prevent fur from getting into the oral cavity.</li>
		<li>Cut the corners of the mouth along the cheek using dissecting scissors to open the oral cavity. Dissect the tongue with mandible (Figure 1B1) and place it in a plastic dish with a layer of plastic wrap.</li>
		<li>Using surgical forceps to hold the tongue under a dissecting microscope, inject the enzyme mixture of dispase (2.5 mg/mL) and collagenase (1.0 mg/mL) in the sub-epithelial space of tongue through the cutting edge (Figure 1B1, arrows) of the posterior tongue.
		<ol>
			<li>Inject 1 mL of enzyme mixture evenly to the whole tongue for tissue collection from both anterior and posterior tongue.</li>
			<li>Inject 0.5 mL of enzyme mixture locally to the anterior tongue for tissue collection from tongue tip or to the posterior tongue for circumvallate papilla tissue collection. 
			&#8203;NOTE: The tongue will swell as the enzyme accumulates (Figure 1B2). Gentle injection of the enzyme can prevent pressure from damaging the epithelium and keep as much enzyme as possible in the tongue.</li>
		</ol>
		</li>
		<li>Wrap the tongue with plastic wrap and incubate the tongue for 30 min at 37 &#176;C.</li>
		<li>Use mini-scissors to dissect the tongue tip and/or circumvallate papilla, and use spatula and fine forceps to transfer tissue to 15 mL of fresh sterile Tyrode&#39;s solution in a 100 mm culture dish.</li>
		<li>Separate the epithelium from the underlying connective tissue in the enzyme-digested sub-epithelial space using mini-scissors. Trim the tissues to a proper size according to the requirement of downstream experiments.</li>
		<li>Wash the separated epithelium and underlying connective tissue twice in 15 mL of fresh sterile Tyrode&#39;s solution in a 100 mm culture dish. 
		&#8203;NOTE: The activities of dispase and collagenase will be inhibited by EDTA in the cell dissociation procedure (step 4.1).</li>
	</ol>
	</li>
</ol>
4. Cell dissociation
NOTE: The cell dissociation protocol described here can be applied to the tongue epithelium and mesenchyme/connective tissue in both E12.5 embryonic and 8-week-old mice. To reduce the cell loss during agitation and transfer of cell suspension, use commercial low retention pipette tips or pre-coated pipette tips with 2.5% BSA in 0.1 M PBS at pH 7.48.
<ol>
	<li>Transfer the tissues using spatula and fine forceps to 3 mL of 0.25% trypsin-EDTA in a new 35 mm culture dish for 30 min at 37 &#176;C. Gently agitate tissues every 5 min with 1 mL pipette tips. 
	NOTE: Do not cut the pipette tip, as the cutting edge can physically damage the dissociated cells.</li>
	<li>Add 500 &#181;L of 5% FBS in DMEM/F12 to stop the reaction and transfer the medium to a 5 mL low binding centrifuge tube.</li>
	<li>Centrifuge cell suspension at 200 x g for 8 min at room temperature and remove the supernatant.</li>
	<li>Gently re-suspend cells in 3 mL of DMEM/F12 containing 10% FBS and 1% BSA using 1 mL pipette tips and filter the cells using a 70 &#181;m cell strainer, followed by a 35 &#181;m cell strainer.</li>
	<li>Centrifuge cell suspension at 200 x g for 8 min at room temperature. Remove most of the medium and leave 50-300 &#181;L as the final volume to re-suspend cells. 
	&#8203;NOTE: Adjust the concentration of cells according to requirements of downstream experiments by changing the final volume of single cell suspension.</li>
</ol>
5. Cell counting and viability test using hemocytometer
NOTE: To improve measurement accuracy, 3 technical replicates are recommended for each sample.
<ol>
	<li>Gently mix 5-10 &#181;L of the single cell suspension with an equal volume of Trypan blue and add onto hemocytometer. 
	NOTE: Clean the hemocytometer thoroughly using 70% ethanol before use. Dusts particles on the hemocytometer will be stained in dark blue and affect the accuracy of viability test. Check the hemocytometer under a microscope.</li>
	<li>Count the total cell number, the number of live cells (white), and the number of dead cells (dark blue) stained by Trypan blue (Figure 2, arrows) respectively in 4 squares with 16 grids (Figure 2) using inverted microscope with imaging system.</li>
	<li>Calculate the cell concentration: 
	Cell concentration = <img alt="Equation 1" src="/files/ftp_upload/62163/62163eq01v2.jpg" />
	<ol>
		<li>Calculate the viability: 
		Viability = <img alt="Equation 2" src="/files/ftp_upload/62163/62163eq02.jpg" /></li>
	</ol>
	</li>
</ol>

<ol>
	<li>Grada, A., Weinbrecht, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Next-generation+sequencing:+methodology+and+application.">Next-generation sequencing: methodology and application.</a> The Journal of investigative dermatology. 133 (8), 11 (2013).</li><li>Whitley, S. K., Horne, W. T., Kolls, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Research+techniques+made+simple:+methodology+and+clinical+applications+of+RNA+sequencing.">Research techniques made simple: methodology and clinical applications of RNA sequencing.</a> Journal of Investigative Dermatology. 136 (8), 77-82 (2016).</li><li>Schaum, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+transcriptomics+of+20+mouse+organs+creates+a+Tabula+Muris:+The+Tabula+Muris+Consortium.">Single-cell transcriptomics of 20 mouse organs creates a Tabula Muris: The Tabula Muris Consortium.</a> Nature. 562 (7727), 367 (2018).</li><li>Sukumaran, S. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole+transcriptome+profiling+of+taste+bud+cells.">Whole transcriptome profiling of taste bud cells.</a> Scientific reports. 7 (1), 1-15 (2017).</li><li>Ren, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptome+analyses+of+taste+organoids+reveal+multiple+pathways+involved+in+taste+cell+generation.">Transcriptome analyses of taste organoids reveal multiple pathways involved in taste cell generation.</a> Scientific Reports. 7 (1), 1-13 (2017).</li><li>Ren, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+Lgr5-or+Lgr6-expressing+taste+stem/progenitor+cells+generate+taste+bud+cells+ex+vivo.">Single Lgr5-or Lgr6-expressing taste stem/progenitor cells generate taste bud cells ex vivo.</a> Proceedings of the National Academy of Sciences. 111 (46), 16401-16406 (2014).</li><li>Venkatesan, N., Boggs, K., Liu, H. -. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Taste+bud+labeling+in+whole+tongue+epithelial+sheet+in+adult+mice.">Taste bud labeling in whole tongue epithelial sheet in adult mice.</a> Tissue Engineering Part C: Methods. 22 (4), 332-337 (2016).</li><li>Nguyen-Ngoc, K. -. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Tissue Morphogenesis. , 135-162 (2015).</li><li>Okubo, T., Clark, C., Hogan, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+lineage+mapping+of+taste+bud+cells+and+keratinocytes+in+the+mouse+tongue+and+soft+palate.">Cell lineage mapping of taste bud cells and keratinocytes in the mouse tongue and soft palate.</a> Stem Cells. 27 (2), 442-450 (2009).</li></ol>

No conflicts of interest declared.

To date, there has not been a detailed protocol available for cell dissociation from the tongue epithelium and underlying mesenchyme/connective tissue. This current cell dissociation protocol provides a reproducible procedure to generate a single cell suspension with a high cell viability (&gt;90%) from mouse tongue tissues, including epithelial sheets and mesenchyme/connective tissues at both embryonic and postnatal stages even though isolated cells from E12.5 and adult mice are different in size. For example, isolated cells from the epithelium and mesenchyme of E12.5 mouse tongues are consistent and small in contrast to a large variability (from small to large) of cells from 8-week-old mouse tongue. With the advantage of the high viability (&gt;90%) of isolated cells, the yielded single cell suspension can facilitate the downstream experiments requiring high quality of viable cells (e.g., single cell RNA sequencing and primary cell cultures).
To guarantee the high viability of cells, careful attention must be paid to several important factors. A proper digestion time with dispase and collagenase mixture can effectively separate the epithelium from mesenchyme/underlying connective tissue while preserving the viable cells. A 20 min incubation for the embryonic tongue and a 30 min incubation for the adult tongue are recommended. Even though epithelial sheets can be easily peeled after enzyme digestion, cutting with scissors for the separation is recommended, which may preserve the stem cell population in the basal area of the tongue epithelium9. The choice of 0.25% trypsin-EDTA over trypsin alone is highly recommended for dissociating cells as 0.25% trypsin-EDTA can dissociate cells more efficiently and keep cells in separation compared to the trypsin alone. The use of cell culture based medium (DMEM/F12 containing 10% FBS and 1% BSA) to re-suspend cells after enzymatic digestion is crucial and contributes to the higher viability of isolated cells compared to cell suspension medium (e.g., 1% BSA in 0.1 M PBS). In addition, isolated cells have a higher survival rate in this medium over time: at least 3 h without significant decrease of cell viability. The pipetting technique is another critical factor to ensure a high cell viability. Gently aspirating the supernatant and re-suspending cells using a pipette can reduce extra cell loss and physical damage to cells. 
The concentration of isolated cells in a single cell suspension is another important consideration for downstream experiments. In a total given cell number, cell concentration can be adjusted based on the final volume of resuspended solution. For the first trial when total cell number is unknown, resuspension of isolated cells should start at a low volume. Then, isolated cells can be diluted based on the requirements of downstream experiments. Of note, in our culture medium based solution, cellular aggregates were found when the concentration was higher than 4000 cells/&#181;L (around 200 cells per square in hemocytometer). The optimal concentrations range from 500 to 2500 cells/&#181;L.

The mammalian tongue is a complex organ critical for taste, speaking, and food processing. It is comprised of multiple types of highly organized tissues compartmentalized by mesenchyme/connective tissue and covered by a stratified epithelial sheet containing taste papillae and taste buds. Cell populations in both tongue epithelium and mesenchyme/connective tissue are heterogeneous. To better understand the functions and distribution of a particular type of cells in the tongue, studies using dissociated cells are necessary. For example, single cell RNA sequencing is a powerful and high-throughput method for transcriptomic profiling in individual cells, which is designed to understand the transcriptome of complex tissue at a single-cell resolution1,2,3,4. Primary cell culture has been proven to be a useful tool to study the function and differentiation of stem/progenitor cells for taste buds5,6. These studies require a large quantity of high-quality isolated cell populations (e.g., sufficient total cell number with proper concentration and high viability).
Thus, there is a need to isolate cells from different regions of the lingual tissues and at different developmental stages. Currently, there is not a detailed protocol available for cell dissociation from the tongue epithelium and underlying mesenchyme/connective tissue. Here, we report an optimized cell dissociation method to prepare cells for experiments requiring a high quality of live cells such as for single cell RNA sequencing and primary stem cell cultures. We found that selection of enzymatic digestion buffer, gentle pipetting, selection of resuspension medium, and optimal centrifugation time and speed are crucial to generate these large quantities of high-quality cells.

<table><tbody><tr><td>bovine serum albumin (BSA)</td><td>Gold Biotechnology</td><td>A-420-100</td><td></td></tr><tr><td>C57BL/6 mouse (C57BL/6J)</td><td>The Jackson Laboratory</td><td>000664</td><td></td></tr><tr><td>collagenase (Collagenase A)</td><td>Sigma-Aldrich</td><td>10103586001</td><td></td></tr><tr><td>culture dish (35 mm in diameter)</td><td>Genesee Scientific</td><td>32-103G</td><td></td></tr><tr><td>culture dish (100 mm in diameter)</td><td>Genesee Scientific</td><td>32-107G</td><td></td></tr><tr><td>dispase (Dispase II)</td><td>Sigma-Aldrich</td><td>04942078001</td><td></td></tr><tr><td>dissecting scissors (Student Fine Scissors)</td><td>Find Science Tool</td><td>91460-11</td><td></td></tr><tr><td>DMEM/F12</td><td>Gibco</td><td>11320033</td><td></td></tr><tr><td>fetal bovine serum (FBS)</td><td>Hyclone</td><td>C838U82</td><td></td></tr><tr><td>fine forceps (Dumount #3 Forceps)</td><td>Find Science Tool</td><td>11293-00</td><td></td></tr><tr><td>hemocytometer</td><td>Hausser Scientific</td><td>3520</td><td></td></tr><tr><td>inverted microscope with imaging system (EVOS XL Core Cell Imaging System)</td><td>Life Technologies</td><td>AMEX1000</td><td></td></tr><tr><td>low retention pipette tips</td><td>METTLER TOLEDO</td><td>17014342</td><td></td></tr><tr><td>mini-scissors (Evo Spring Scissors)</td><td>Fine Science Tool</td><td>15800-01</td><td></td></tr><tr><td>plastic warp</td><td>VWR</td><td>46610-056</td><td></td></tr><tr><td>spatula (Moria Spoon)</td><td>Fine Science Tool</td><td>10321-08</td><td></td></tr><tr><td>surgical forceps (Dumount #2 Laminectomy Forceps)</td><td>Fine Science Tool</td><td>11223-20</td><td></td></tr><tr><td>Trypan blue</td><td>Gibco</td><td>15250061</td><td></td></tr><tr><td>Tyrode&amp;rsquo;s solution</td><td>Sigma-Aldrich</td><td>T2145-10L</td><td>made from Tyrode&#039;s salts</td></tr><tr><td>0.25% typsin-EDTA</td><td>Gibco</td><td>25200056</td><td></td></tr><tr><td>0.1 M Phosphate-Buffered Saline (PBS)</td><td>Hoefer</td><td>33946</td><td>made from 1 M PBS</td></tr><tr><td>0.22-&amp;mu;m syringe filter</td><td>Genesee Scientific</td><td>25-243</td><td></td></tr><tr><td>70% ethanol</td><td>Koptec</td><td>233919</td><td>made from 100% ethanol</td></tr><tr><td>1-mL syringe</td><td>BD</td><td>8194938</td><td></td></tr><tr><td>5-mL low binding microcentrifuge tube</td><td>Eppendorf</td><td>30122348</td><td></td></tr><tr><td>30-G needle</td><td>BD</td><td>9193532</td><td></td></tr><tr><td>35-&amp;mu;m cell strainer</td><td>Falcon</td><td>64750</td><td></td></tr><tr><td>70-&amp;mu;m cell strainer</td><td>Falcon</td><td>64752</td><td></td></tr></tbody></table>

cell dissociation from the tongue epithelium and mesenchyme/connective tissue of embryonic-day 12.5 and 8-week-old mice

Cell dissociation has been an essential procedure for studies at the individual-cell level and/or at a cell-population level (e.g.,&#160;single cell RNA sequencing and primary cell culture). Yielding viable, healthy cells in large quantities is critical, and the optimal conditions to do so are tissue dependent. Cell populations in the tongue epithelium and underlying mesenchyme/connective tissue are heterogeneous and tissue structures vary in different regions and at different developmental stages. We have tested protocols for isolating cells from the mouse tongue epithelium and mesenchyme/connective tissue in the early developmental [embryonic day 12.5 (E12.5)] and young adult (8-week) stages. A clean separation between the epithelium and underlying mesenchyme/connective tissue was easy to accomplish. However, to further process and isolate cells, yielding viable healthy cells in large quantities, and careful selection of enzymatic digestion buffer, incubation time, and centrifugation speed and time are critical. Incubation of separated epithelium or underlying mesenchyme/connective tissue in 0.25% Trypsin-EDTA for 30 min at 37 &#176;C, followed by centrifugation at 200 x g for 8 min resulted in a high yield of cells at a high viability rate (&#62;90%) regardless of the mouse stages and tongue regions. Moreover, we found that both dissociated epithelial and mesenchymal/connective tissue cells from embryonic and adult tongues could survive in the cell culture-based medium for at least 3 h without a significant decrease of cell viability. The protocols will be useful for studies that require the preparation of isolated cells from mouse tongues at early developmental (E12.5) and young adult (8-week) stages requiring cell dissociation from different tissue compartments.

Separation of the tongue epithelium from the underlying mesenchyme/connective tissue 
In the embryonic mouse tongue, a gap in the sub-epithelial space is visible after proper enzyme digestion. Epithelial sheets of some tongues are separated without mechanical force during the incubation.
In the adult mouse tongue, a successful enzyme injection is indicated by the swelling in the injected areas (Figure 1B2), which suggests that the enzyme can be held by the tongue. Insufficient enzyme and/or deep needle insertion to the mesenchyme and/or tongue epithelial penetration by needle will induce a partial swelling of the injection area or no swelling at all. After enzyme digestion, the underlying connective tissues with proper enzyme digestion become loose and sticky. A gap in the sub-epithelial space is visible when gently lifting the edge of the epithelial sheet.
Effect of cell dissociation on total cell number and viability 
With step 4, E12.5 tongues, the epithelial sheets, and thin layers of mesenchyme immediately under the epithelium of tongues were pooled, respectively. Manual cell counting using a hemocytometer (Figure 2) demonstrated that the protocol yielded 63,917 cells in total with a viability of 95.2% from the epithelial sheets (around 0.3 mm2 in size per tongue) (Figure 1A3), and 294,333 cells in total with a viability of 96.3% from the mesenchyme (around 0.3 mm2 in size per tongue) (Figure 1A4).
Using 10 adult tongues at 8 weeks of age, the pieces of the epithelial sheets of the tongue tip (where taste buds are densely distributed), epithelial sheets of circumvallate papillae, and thin layers of connective tissue immediately under the epithelium of circumvallate papillae were pooled, respectively. A manual cell count using the hemocytometer (Figure 2) demonstrated that the protocol yielded 187,333 cells in total with a viability of 95.4% from epithelial sheets of tongue tip (around 0.075 mm2 in size per tongue) (Figure 1B3), 544,000 cells in total with a viability of 96.3% from epithelial sheets of circumvallate papillae (around 0.1 mm2 in size per tongue) (Figure 1B5), and 150,500 cells in total with a viability of 93% from connective tissues (around 0.1 mm2 in size per tongue) (Figure 1B6).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62163/62163fig01.jpg" /> 
Figure 1. Tissue preparation for cell dissociation. A) Representative images of an E12.5 whole embryo (A1), dorsal view of the dissected tongue (A2), and epithelial sheets (A3) and mesenchyme (A4) separated from tongues. B) Representative images of an adult tongue before (B1) and after enzyme injection (B2). Dorsal view of an epithelial sheet (B3) and mesenchyme (B4) of tongue tip, and an epithelial sheet (B5) and underlying connective tissue (B6)&#160;of circumvallate papilla. Scale bars: 1 mm in A1, A3, A4, B1, B2; 200 &#181;m in A2; 100 &#181;m in B3, B4, B5, and B6. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62163/62163fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62163/62163fig02.jpg" /> 
Figure 2. Representative images of isolated cells visualized in hemocytometer. A) Isolated cells from epithelial sheets (A1) and mesenchyme (A2) of embryos at E12.5. B) Isolated cells from epithelial sheets (B1) and underlying connective tissue cores (B2) of circumvallate papillae at 8 weeks of age. Dashd lines encircle the grids amplified in top right corner. Arrows point to dead cells stained by Trypan blue. Scale bars: 100 &#181;m for B1,B2, B3, and B4; 25 &#181;m for high power images in top right corner. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62163/62163fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Cell Dissociation from the Tongue Epithelium and Mesenchyme/Connective Tissue of Embryonic-Day 12.5 and 8-Week-Old Mice at JoVE.com

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

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 has been an essential procedure for studies at the individual-cell level and/or at a cell-population level (e.g.,&#160;single cell RNA ...

cell-dissociation-from-tongue-epithelium-mesenchymeconnective-tissue

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

4.4K Views.  University of Georgia. 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. 

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

Fabrication of Tongue Extracellular Matrix and Reconstitution of Tongue Squamous Cell Carcinoma In Vitro

In order to construct an effective and realistic model for tongue squamous cell carcinoma (TSCC) in vitro, the methods were created to produce decellularized tongue extracellular matrix (TEM) which provides functional scaffolds for TSCC construction. TEM provides an in vitro niche for cell growth, differentiation, and cell migration. The microstructures of native extracellular matrix (ECM) and biochemical compositions retained in the decellularized matrix provide tissue-specific niches for anchoring cells. The fabrication of TEM can be realized by deoxyribonuclease (DNase) digestion accompanied with a serious of organic or inorganic pretreatment. This protocol is easy to operate and ensures high efficiency for the decellularization. The TEM showed favorable cytocompatibility for TSCC cells under static or stirred culture conditions, which enables the construction of the TSCC model. A self-made bioreactor was also used for the persistent stirred condition for cell culture. Reconstructed TSCC using TEM showed the characteristics and properties resembling clinical TSCC histopathology, suggesting the potential in TSCC research.

Fabrication of Tongue Extracellular Matrix and Reconstitution of Tongue Squamous Cell Carcinoma In Vitro

A method is shown here for the preparation of the tongue extracellular matrix (TEM) with efficient decellularization. The TEM can be used as functional scaffolds for the reconstruction of a tongue squamous cell carcinoma (TSCC) model under static or stirred culture conditions.

In order to construct an effective and realistic model for tongue squamous cell carcinoma (TSCC) in vitro, the methods were created to produce ...

Cancer Research

Modeling Oral-Esophageal Squamous Cell Carcinoma in 3D Organoids

Esophageal squamous cell carcinoma (ESCC) is prevalent worldwide, accounting for 90% of all esophageal cancer cases each year, and is the deadliest of all human squamous cell carcinomas. Despite recent progress in defining the molecular changes accompanying ESCC initiation and development, patient prognosis remains poor. The functional annotation of these molecular changes is the necessary next step and requires models that both capture the molecular features of ESCC and can be readily and inexpensively manipulated for functional annotation. Mice treated with the tobacco smoke mimetic 4-nitroquinoline 1-oxide (4NQO) predictably form ESCC and esophageal preneoplasia. Of note, 4NQO lesions also arise in the oral cavity, most commonly in the tongue, as well as the forestomach, which all share the stratified squamous epithelium. However, these mice cannot be simply manipulated for functional hypothesis testing, as generating isogenic mouse models is time- and resource-intensive. Herein, we overcome this limitation by generating single cell-derived three-dimensional (3D) organoids from mice treated with 4NQO to characterize murine ESCC or preneoplastic cells ex vivo. These organoids capture the salient features of ESCC and esophageal preneoplasia, can be cheaply and quickly leveraged to form isogenic models, and can be utilized for syngeneic transplantation experiments. We demonstrate how to generate 3D organoids from normal, preneoplastic, and SCC murine esophageal tissue and maintain and cryopreserve these organoids. The applications of these versatile organoids are broad and include the utilization of genetically engineered mice and further characterization by flow cytometry or immunohistochemistry, the generation of isogeneic organoid lines using CRISPR technologies, and drug screening or syngeneic transplantation. We believe that the widespread adoption of the techniques demonstrated in this protocol will accelerate progress in this field to combat the severe burden of ESCC.

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.

Esophageal squamous cell carcinoma (ESCC) is prevalent worldwide, accounting for 90% of all esophageal cancer cases each year, and is the deadliest of all ...

Isolation and Time-Lapse Imaging of Primary Mouse Embryonic Palatal Mesenchyme Cells to Analyze Collective Movement Attributes

Development of the palate is a dynamic process, which involves vertical growth of bilateral palatal shelves next to the tongue followed by elevation and fusion above the tongue. Defects in this process lead to cleft palate, a common birth defect. Recent studies have shown that palatal shelf elevation involves a remodeling process that transforms the orientation of the shelf from a vertical to a horizontal one. The role of the palatal shelf mesenchymal cells in this dynamic remodeling has been difficult to study. Time-lapse-imaging-based quantitative analysis has been recently used to show that primary mouse embryonic palatal mesenchymal (MEPM) cells can self-organize into a collective movement. Quantitative analyses could identify differences in mutant MEPM cells from a mouse model with palate elevation defects. This paper describes methods to isolate and culture MEPM cells from E13.5 embryos-specifically for time-lapse imaging-and to determine various cellular attributes of collective movement, including measures for stream formation, shape alignment, and persistence of direction. It posits that MEPM cells can serve as a proxy model for studying the role of palatal shelf mesenchyme during the dynamic process of elevation. These quantitative methods will allow investigators in the craniofacial field to assess and compare collective movement attributes in control and mutant cells, which will augment the understanding of mesenchymal remodeling during palatal shelf elevation. Furthermore, MEPM cells provide a rare mesenchymal cell model for investigation of collective cell movement in general.

We present a protocol for isolation and culture of primary mouse embryonic palatal mesenchymal cells for time-lapse imaging of two-dimensional (2D) growth and wound-repair assays. We also provide the methodology for analysis of the time-lapse imaging data to determine cell-stream formation and directional motility.

Development of the palate is a dynamic process, which involves vertical growth of bilateral palatal shelves next to the tongue followed by elevation and ...

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.

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 ...

Separation of Mouse Embryonic Facial Ectoderm and Mesenchyme

Orofacial clefts are the most frequent craniofacial defects, which affect 1.5 in 1,000 newborns worldwide1,2. Orofacial clefting is caused by abnormal facial development3. In human and mouse, initial growth and patterning of the face relies on several small buds of tissue, the facial prominences4,5. The face is derived from six main prominences: paired frontal nasal processes (FNP), maxillary prominences (MxP) and mandibular prominences (MdP). These prominences consist of swellings of mesenchyme that are encased in an overlying epithelium. Studies in multiple species have shown that signaling crosstalk between facial ectoderm and mesenchyme is critical for shaping the face6. Yet, mechanistic details concerning the genes involved in these signaling relays are lacking. One way to gain a comprehensive understanding of gene expression, transcription factor binding, and chromatin marks associated with the developing facial ectoderm and mesenchyme is to isolate and characterize the separated tissue compartments.
Here we present a method for separating facial ectoderm and mesenchyme at embryonic day (E) 10.5, a critical developmental stage in mouse facial formation that precedes fusion of the prominences. Our method is adapted from the approach we have previously used for dissecting facial prominences7. In this earlier study we had employed inbred C57BL/6 mice as this strain has become a standard for genetics, genomics and facial morphology8. Here, though, due to the more limited quantities of tissue available, we have utilized the outbred CD-1 strain that is cheaper to purchase, more robust for husbandry, and tending to produce more embryos (12-18) per litter than any inbred mouse strain8. Following embryo isolation, neutral protease Dispase II was used to treat the whole embryo. Then, the facial prominences were dissected out, and the facial ectoderm was separated from the mesenchyme. This method keeps both the facial ectoderm and mesenchyme intact. The samples obtained using this methodology can be used for techniques including protein detection, chromatin immunoprecipitation (ChIP) assay, microarray studies, and RNA-seq.

A protocol for separation of embryo facial ectoderm and mesenchyme is described. We use Dispase II to treat whole embryos first, dissect whole facial prominences out, and then separate the facial ectoderm and mesenchyme.

 Orofacial clefts are the most frequent  craniofacial defects, which affect 1.5 in 1,000 newborns worldwide1,2. Orofacial clefting is caused by abnormal ...

Biology

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

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse incisor provides a model for these processes. This remarkable organ grows continuously throughout the animal&#39;s life and generates all the necessary cell types from active pools of adult stem cells housed in the labial (toward the lip) and lingual (toward the tongue) cervical loop (CL) regions. Only the dental stem cells from the labial CL give rise to ameloblasts that generate enamel, the outer covering of teeth, on the labial surface. This asymmetric enamel formation allows abrasion at the incisor tip, and progenitors and stem cells in the proximal incisor ensure that the dental tissues are constantly replenished. The ability to isolate and grow these progenitor or stem cells in vitro allows their expansion and opens doors to numerous experiments not achievable in vivo, such as high throughput testing of potential stem cell regulatory factors. Here, we describe and demonstrate a reliable and consistent method to culture cells from the labial CL of the mouse incisor.

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.

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse ...

Generation and Characterization of Murine Oral Mucosal Organoid Cultures

The mucous lining covering the inside of our mouth, the oral mucosa, is a highly compartmentalized tissue and can be subdivided into the buccal mucosa, gingiva, lips, palate, and tongue. Its uppermost layer, the oral epithelium, is maintained by adult stem cells throughout life. Proliferation and differentiation of adult epithelial stem cells have been intensively studied using in vivo mouse models as well as two-dimensional (2D) feeder-cell based in vitro models. Complementary to these methods is organoid technology, where adult stem cells are embedded into an extracellular matrix (ECM)-rich hydrogel and provided with a culture medium containing a defined cocktail of growth factors. Under these conditions, adult stem cells proliferate and spontaneously form three-dimensional (3D) cell clusters, the so-called organoids. Organoid cultures were initially established from murine small intestinal epithelial stem cells. However, the method has since been adapted for other epithelial stem cell types. Here, we describe a protocol for the generation and characterization of murine oral mucosal organoid cultures. Primary epithelial cells are isolated from murine tongue tissue, embedded into an ECM hydrogel, and cultured in a medium containing: epidermal growth factor (EGF), R-spondin, and fibroblast growth factor (FGF) 10. Within 7 to 14 days of initial seeding, the resulting organoids can be passaged for further expansion and cryopreservation. We additionally present strategies for the characterization of established organoid cultures via 3D whole-mount imaging and gene-expression analysis. This protocol may serve as a tool to investigate oral epithelial stem cell behavior ex vivo in a reductionist manner.

We present a method for the generation and characterization of oral mucosal organoid cultures derived from the tongue epithelium of adult mice.

The mucous lining covering the inside of our mouth, the oral mucosa, is a highly compartmentalized tissue and can be subdivided into the buccal mucosa, ...

Isolation of Circumvallate Papillae (CVP) Epithelium from Mouse Model: An Enzymatic Procedure to Separate CVP Epithelium from Mouse Tongue

Source: Shechtman, L. A. et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/video/62300" target="_blank">Generation and Culture of Lingual Organoids Derived from Adult Mouse Taste Stem Cells</a>. J. Vis. Exp. (2021)
In this video, we describe the isolation of circumvallate papillae (CVP) epithelium from a murine tongue.

Source: Shechtman, L. A. et al. Generation and Culture of Lingual Organoids Derived from Adult Mouse Taste Stem Cells. J. Vis. Exp. (2021)
In this video, ...

JoVE Encyclopedia of Experiments

Rodent Models

Adult

Fabrication of Extracellular Matrix from Mouse Model: An Effective Procedure to Prepare Decellularized Tongue Extracellular Matrix from Murine Tongue

Source: Yao, Y. et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/video/57235" target="_blank">Fabrication of Tongue Extracellular Matrix and Reconstitution of Tongue Squamous Cell Carcinoma In Vitro.</a> J. Vis. Exp. (2018)
This video demonstrates the preparation of the murine tongue extracellular matrix by using the decellularization method, which involves exposing the harvested tongue to a series of physical, chemical, and enzymatic treatments. The resulting TEM scaffold retains its native extracellular matrix composition, removing the cellular components.

Source: Yao, Y. et al. Fabrication of Tongue Extracellular Matrix and Reconstitution of Tongue Squamous Cell Carcinoma In Vitro. J. Vis. Exp. (2018)
This ...

Head and Neck Cancer

Assays & techniques

Isolation and Culturing of Cells from Mouse Embryo Dorsolateral Telencephalons

Source: Landeira, B. S., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/56063">Live Imaging of Primary Cerebral Cortex Cells Using a 2D Culture System.</a> J. Vis. Exp. (2017)
This video demonstrates a protocol for isolating and culturing primary cerebral cortex cells from the dorsolateral telencephalon of mouse embryos.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

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

Summary

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Chapters in this video

Fabrication of Tongue Extracellular Matrix and Reconstitution of Tongue Squamous Cell Carcinoma In Vitro

Modeling Oral-Esophageal Squamous Cell Carcinoma in 3D Organoids

Isolation and Time-Lapse Imaging of Primary Mouse Embryonic Palatal Mesenchyme Cells to Analyze Collective Movement Attributes

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

Separation of Mouse Embryonic Facial Ectoderm and Mesenchyme

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

Generation and Characterization of Murine Oral Mucosal Organoid Cultures

Isolation of Circumvallate Papillae (CVP) Epithelium from Mouse Model: An Enzymatic Procedure to Separate CVP Epithelium from Mouse Tongue

Fabrication of Extracellular Matrix from Mouse Model: An Effective Procedure to Prepare Decellularized Tongue Extracellular Matrix from Murine Tongue

Isolation and Culturing of Cells from Mouse Embryo Dorsolateral Telencephalons