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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#8217;s solution</td>
			<td>Sigma-Aldrich</td>
			<td>T2145-10L</td>
			<td>made from Tyrode&#39;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-&#956;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-&#956;m cell strainer</td>
			<td>Falcon</td>
			<td>64750</td>
			<td></td>
		</tr>
		<tr>
			<td>70-&#956;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 ...

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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

Efficient Derivation of Retinal Pigment Epithelium Cells from Stem Cells

No cure has been discovered for age-related macular degeneration (AMD), the leading cause of vision loss in people over the age of 55. AMD is complex multifactorial disease with an unknown etiology, although it is largely thought to occur due to death or dysfunction of the retinal pigment epithelium (RPE), a monolayer of cells that underlies the retina and provides critical support for photoreceptors. RPE cell replacement strategies may hold great promise for providing therapeutic relief for a large subset of AMD patients, and RPE cells that strongly resemble primary human cells (hRPE) have been generated in multiple independent labs, including our own. In addition, the uses for iPS-RPE are not limited to cell-based therapies, but also have been used to model RPE diseases. These types of studies may not only elucidate the molecular bases of the diseases, but also serve as invaluable tools for developing and testing novel drugs. We present here an optimized protocol for directed differentiation of RPE from stem cells. Adding nicotinamide and either Activin A or IDE-1, a small molecule that mimics its effects, at specific time points, greatly enhances the yield of RPE cells. Using this technique we can derive large numbers of low passage RPE in as early as three months.

Stem cell-derived retinal pigment epithelium (RPE) cells may be used for multiple applications including cell-based therapies for retinal degeneration, disease modeling, and drug studies. Here we present a simple protocol for reproducibly deriving RPE from stem cells.

No cure has been discovered for age-related macular degeneration  (AMD), the leading cause of vision loss in people over the age of 55. AMD is complex ...

Assaying Blood Cell Populations of the Drosophila melanogaster Larva

In vertebrates, hematopoiesis is regulated by inductive microenvironments (niches). Likewise, in the invertebrate model organism&#160;Drosophila melanogaster, inductive microenvironments known as larval Hematopoietic Pockets (HPs) have been identified as anatomical sites for the development and regulation of blood cells (hemocytes), in particular of the self-renewing macrophage lineage.&#160;HPs are segmentally repeated pockets between the epidermis and muscle layers of the larva, which also comprise sensory neurons of the peripheral nervous system. In the larva, resident (sessile) hemocytes are exposed to anti-apoptotic, adhesive and proliferative cues from these sensory neurons and potentially other components of the HPs, such as the lining muscle and epithelial layers. During normal development, gradual release of resident hemocytes from the HPs fuels the population of circulating hemocytes, which culminates in the release of most of the resident hemocytes at the beginning of metamorphosis. Immune assaults, physical injury or mechanical disturbance trigger the premature release of resident hemocytes into circulation. The switch of larval hemocytes between resident locations and circulation raises the need for a common standard/procedure to selectively isolate and quantify these two populations of blood cells from single Drosophila larvae. Accordingly, this protocol describes an automated method to release and quantify the resident and circulating hemocytes from single larvae. The method facilitates ex vivo approaches, and may be adapted to serve a variety of developmental stages of Drosophila and other invertebrate organisms.

Assaying Blood Cell Populations of the Drosophila melanogaster Larva

Drosophila blood cells, or hemocytes, cycle between resident sites and circulation. In the larva, resident (sessile) hemocytes localize to inductive microenvironments, the Hematopoietic Pockets, while circulating hemocytes move freely in the hemolymph. The goal of this protocol is the standardized isolation and quantification of these two, behaviorally distinct but interchanging, hemocyte populations.

In vertebrates, hematopoiesis is regulated by inductive microenvironments (niches). Likewise, in the invertebrate model organism&#160;Drosophila ...

Isolation and Expansion of Mesenchymal Stem/Stromal Cells Derived from Human Placenta Tissue

Mesenchymal stem/stromal cells (MSC) are promising candidates for use in cell-based therapies. In most cases, therapeutic response appears to be cell-dose dependent. Human term placenta is rich in MSC and is a physically large tissue that is generally discarded following birth. Placenta is an ideal starting material for the large-scale manufacture of multiple cell doses of allogeneic MSC. The placenta is a fetomaternal organ from which either fetal or maternal tissue can be isolated. This article describes the placental anatomy and procedure to dissect apart the decidua (maternal), chorionic villi (fetal), and chorionic plate (fetal) tissue. The protocol then outlines how to isolate MSC from each dissected tissue region, and provides representative analysis of expanded MSC derived from the respective tissue types. These methods are intended for pre-clinical MSC isolation, but have also been adapted for clinical manufacture of placental MSC for human therapeutic use.

Herein we describe methods for the dissection of fetal and maternal tissues from human term placenta, followed by isolation and expansion of mesenchymal stem/stromal cells (MSC) from these tissues.

Mesenchymal stem/stromal cells (MSC) are promising candidates for use in cell-based therapies. In most cases, therapeutic response appears to be cell-dose ...

Isolation of Perivascular Multipotent Precursor Cell Populations from Human Cardiac Tissue

Multipotent mesenchymal stem/stromal cells (MSC) were conventionally isolated, through their plastic adherence, from primary tissue digests whilst their anatomical tissue location remained unclear. The recent discovery of defined perivascular and MSC cell marker expression by perivascular cells in multiple tissues by our group and other researchers has provided an opportunity to prospectively isolate and purify specific homogenous subpopulations of multipotent perivascular precursor cells. We have previously demonstrated the use of fluorescent activated cell sorting (FACS) to purify microvascular CD146+CD34- pericytes and vascular CD34+CD146- adventitial cells from human skeletal muscle. Herein we describe a method to simultaneously isolate these two perivascular cell subsets from human myocardium by FACS, based on the expression of a defined set of cell surface markers for positive and negative selections. This method thus makes available two specific subpopulations of multipotent cardiac MSC-like precursor cells for use in basic research and/or therapeutic investigations.

Human cardiac tissue harbours multipotent perivascular precursor cell populations that may be suitable for myocardial regeneration. The technique described here allows for the simultaneous isolation and purification of two multipotent stromal cell populations associated with native blood vessels, i.e. CD146+CD34- pericytes and CD34+CD146- adventitial cells, from the human myocardium.

Multipotent mesenchymal stem/stromal cells (MSC) were conventionally isolated, through their plastic adherence, from primary tissue digests whilst their ...

Using In Vivo and Tissue and Cell Explant Approaches to Study the Morphogenesis and Pathogenesis of the Embryonic and Perinatal Aorta

The aorta is the largest artery in the body. The aortic wall is composed of an inner layer of endothelial cells, a middle layer of alternating elastic lamellae and smooth muscle cells (SMCs), and an outer layer of fibroblasts and extracellular matrix. In contrast to the widespread study of pathological models (e.g., atherosclerosis) in the adult aorta, much less is known about the embryonic and perinatal aorta. Here, we focus on SMCs and provide protocols for the analysis of the morphogenesis and pathogenesis of embryonic and perinatal aortic SMCs in normal development and disease. Specifically, the four protocols included are: i) in vivo embryonic fate mapping and clonal analysis; ii) explant embryonic aorta culture; iii) SMC isolation from the perinatal aorta; and iv) subcutaneous osmotic mini-pump placement in pregnant (or non-pregnant) mice. Thus, these approaches facilitate the investigation of the origin(s), fate, and clonal architecture of SMCs in the aorta in vivo. They allow for modulating embryonic aorta morphogenesis in utero by continuous exposure to pharmacological agents. In addition, isolated aortic tissue explants or aortic SMCs can be used to gain insights into the role of specific gene targets during fundamental processes such as muscularization, proliferation, and migration. These hypothesis-generating experiments on isolated SMCs and the explanted aorta can then be assessed in the in vivo context through pharmacological and genetic approaches.

Using In Vivo and Tissue and Cell Explant Approaches to Study the Morphogenesis and Pathogenesis of the Embryonic and Perinatal Aorta

Protocols for studying the embryonic and perinatal murine aorta using in vivo clonal analysis and fate mapping, aortic explants, and isolated smooth muscle cells are detailed here. These diverse approaches facilitate the investigation of the morphogenesis of the embryonic and perinatal aorta in normal development and the pathogenesis in disease.

The aorta is the largest artery in the body. The aortic wall is composed of an inner layer of endothelial cells, a middle layer of alternating elastic ...

Live Imaging of Mouse Secondary Palate Fusion

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to cleft secondary palate, a common congenital anomaly in humans. Secondary palate fusion has been extensively studied leading to several proposed cellular mechanisms that may mediate this process. However, these studies have been mostly performed on fixed embryonic tissues at progressive timepoints during development or in fixed explant cultures analyzed at static timepoints. Static analysis is limited for the analysis of dynamic morphogenetic processes such a palate fusion and what types of dynamic cellular behaviors mediate palatal fusion is incompletely understood. Here we describe a protocol for live imaging of ex vivo secondary palate fusion in mouse embryos. To examine cellular behaviors of palate fusion, epithelial-specific Keratin14-cre was used to label palate epithelial cells in ROSA26-mTmGflox reporter embryos. To visualize filamentous actin, Lifeact-mRFPruby reporter mice were used. Live imaging of secondary palate fusion was performed by dissecting recently-adhered secondary palatal shelves of embryonic day (E) 14.5 stage embryos and culturing in agarose-containing media on a glass bottom dish to enable imaging with an inverted confocal microscope. Using this method, we have detected a variety of novel cellular behaviors during secondary palate fusion. An appreciation of how distinct cell behaviors are coordinated in space and time greatly contributes to our understanding of this dynamic morphogenetic process. This protocol can be applied to mutant mouse lines, or cultures treated with pharmacological inhibitors to further advance understanding of how secondary palate fusion is controlled.

Here, we present a protocol for live imaging of mouse secondary palate fusion using confocal microscopy. This protocol can be used in combination with a variety of fluorescent reporter mouse lines, and with pathway inhibitors for mechanistic insight. This protocol can be adapted for live imaging in other developmental systems.

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to ...

A Simplified and Efficient Method to Isolate Primary Human Keratinocytes from Adult Skin Tissue

Primary human keratinocytes isolated from fresh skin tissues and their expansion in vitro have been widely used for laboratory research and for clinical applications. The conventional isolation method of human keratinocytes involves a two-step sequential enzymatic digestion procedure, which has been proven to be inefficient in generating primary cells from adult tissues due to the low cell recovery rate and reduced cell viability. We recently reported an advanced method to isolate human primary epidermal progenitor cells from skin tissues that utilizes the Rho kinase inhibitor Y-27632 in the medium. Compared with the traditional protocol, this new method is simpler, easier, and less time-consuming, and increases epithelial stem cell yield and enhances their stem cell characteristics. Moreover, the new methodology does not require the separation of the epidermis from the dermis, and, therefore, is suitable for isolating cells from different types of adult tissues. This new isolation method overcomes the major shortcomings of conventional methods and is more suitable for producing large numbers of epidermal cells with high potency both for laboratory and for clinical applications. Here, we describe the new method in detail.

Here we present a protocol to efficiently isolate primary human keratinocytes from adult skin tissues. This method simplifies the conventional procedure by using the ROCK Inhibitor Y-27632 in the inoculation medium to spontaneously separate epidermal cells from dermal cells.

Primary human keratinocytes isolated from fresh skin tissues and their expansion in vitro have been widely used for laboratory research and for clinical ...

Engineering Transplantation-suitable Retinal Pigment Epithelium Tissue Derived from Human Embryonic Stem Cells

Several pathological conditions of the eye affect the functionality and/or the survival of the retinal pigment epithelium (RPE). These include some forms of retinitis pigmentosa (RP) and age-related macular degeneration (AMD). Cell therapy is one of the most promising therapeutic strategies proposed to cure these diseases, with already encouraging preliminary results in humans. However, the method of preparation of the graft has a significant impact on its functional outcomes in vivo. Indeed, RPE cells grafted as a cell suspension are less functional than the same cells transplanted as a retinal tissue. Herein, we describe a simple and reproducible method to engineer RPE tissue and its preparation for an in vivo implantation. RPE cells derived from human pluripotent stem cells are seeded on a biological support, the human amniotic membrane (hAM). Compared to artificial scaffolds, this support has the advantage of having a basement membrane that is close to the Bruch&#39;s membrane where endogenous RPE cells are attached. However, its manipulation is not easy, and we developed several strategies for its proper culturing and preparation for grafting in vivo.

We describe a method to engineer a retinal tissue composed of retinal pigment epithelial cells derived from human pluripotent stem cells cultured on top of human amniotic membranes and its preparation for grafting in animal models.

Several pathological conditions of the eye affect the functionality and/or the survival of the retinal pigment epithelium (RPE). These include some forms ...

Generation and Expansion of Human Cardiomyocytes from Patient Peripheral Blood Mononuclear Cells

Generating patient-specific cardiomyocytes from a single blood draw has attracted tremendous interest in precision medicine on cardiovascular disease. Cardiac differentiation from human induced pluripotent stem cells (iPSCs) is modulated by defined signaling pathways that are essential for embryonic heart development. Numerous cardiac differentiation methods on 2-D and 3-D platforms have been developed with various efficiencies and cardiomyocyte yield. This has puzzled investigators outside the field as the variety of these methods can be difficult to follow. Here we present a comprehensive protocol that elaborates robust generation and expansion of patient-specific cardiomyocytes from peripheral blood mononuclear cells (PBMCs). We first describe a high-efficiency iPSC reprogramming protocol from a patient&#39;s blood sample using non-integration Sendai virus vectors. We then detail a small molecule-mediated monolayer differentiation method that can robustly produce beating cardiomyocytes from most human iPSC lines. In addition, a scalable cardiomyocyte expansion protocol is introduced using a small molecule (CHIR99021) that could rapidly expand patient-derived cardiomyocytes for industrial- and clinical-grade applications. At the end, detailed protocols for molecular identification and electrophysiological characterization of these iPSC-CMs are depicted. We expect this protocol to be pragmatic for beginners with limited knowledge on cardiovascular development and stem cell biology.

Here, we present a protocol to robustly generate and expand human cardiomyocytes from patient peripheral blood mononuclear cells.

Generating patient-specific cardiomyocytes from a single blood draw has attracted tremendous interest in precision medicine on cardiovascular disease. ...

Murine Neural Plate Targeting by In Utero Nano-Injection (NEPTUNE) at Embryonic Day 7.5

Manipulating gene expression in the developing mouse brain in utero holds great potential for functional genetics studies. However, it has previously largely been restricted to the manipulation of embryonic stages post-neurulation. A protocol was developed to inject the amniotic cavity at embryonic day (E)7.5 and deliver lentivirus, encoding cDNA or shRNA, targeting &gt;95% of the neural plate and neural crest cells, contributing to the future brain, spinal cord, and peripheral nervous system. This protocol describes the steps necessary to achieve successful transduction, including grinding of the glass capillary needles, pregnancy verification, developmental staging using ultrasound imaging, and optimal injection volumes matched to embryonic stages. 
Following this protocol, it is possible to achieve transduction of &gt;95% of the developing brain with high-titer lentivirus and thus perform whole-brain genetic manipulation. In contrast, it is possible to achieve mosaic transduction using lower viral titers, allowing for genetic screening or lineage tracing. Injection at E7.5 also targets ectoderm and neural crest contributing to distinct compartments of the eye, tongue, and peripheral nervous system. This technique thus offers the possibility to manipulate gene expression in mouse neural-plate- and ectoderm-derived tissues from preneurulation stages, with the benefit of reducing the number of mice used in experiments.

Murine Neural Plate Targeting by In Utero Nano-Injection NEPTUNE at Embryonic Day 7.5

In this protocol, we describe how to inject the mouse amniotic cavity at E7.5 with lentivirus, leading to uniform transduction of the entire neural plate, with minimal detrimental effects on survival or embryonic development.

Manipulating gene expression in the developing mouse brain in utero holds great potential for functional genetics studies. However, it has previously ...

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

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Efficient Derivation of Retinal Pigment Epithelium Cells from Stem Cells

Assaying Blood Cell Populations of the Drosophila melanogaster Larva

Isolation and Expansion of Mesenchymal Stem/Stromal Cells Derived from Human Placenta Tissue

Isolation of Perivascular Multipotent Precursor Cell Populations from Human Cardiac Tissue

Using In Vivo and Tissue and Cell Explant Approaches to Study the Morphogenesis and Pathogenesis of the Embryonic and Perinatal Aorta

Live Imaging of Mouse Secondary Palate Fusion

A Simplified and Efficient Method to Isolate Primary Human Keratinocytes from Adult Skin Tissue

Engineering Transplantation-suitable Retinal Pigment Epithelium Tissue Derived from Human Embryonic Stem Cells

Generation and Expansion of Human Cardiomyocytes from Patient Peripheral Blood Mononuclear Cells

Murine Neural Plate Targeting by In Utero Nano-Injection (NEPTUNE) at Embryonic Day 7.5