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

Summary

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.

Abstract

Cell dissociation has been an essential procedure for studies at the individual-cell level and/or at a cell-population level (e.g., 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 °C, followed by centrifugation at 200 x g for 8 min resulted in a high yield of cells at a high viability rate (>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.

Introduction

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 designe....

Protocol

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 °C under 12-h day/night cycles.

1. 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 experimen....

Results

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.......

Discussion

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 (>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 .......

Materials

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