Name: cell dissociation from the tongue epithelium and mesenchyme/connective tissue of embryonic-day 12.5 and 8-week-old mice
Uploaded: 2021-01-21T15:00:00
Description: 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

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...

<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>

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 ...

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 s...

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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