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., 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.
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.
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.
2. Preparation before experiment
NOTE: The instruments required for this protocol are listed in the Table of Materials.
3. Separation of the tongue epithelium from the mesenchyme/underlying connective tissue
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.
5. Cell counting and viability test using hemocytometer
NOTE: To improve measurement accuracy, 3 technical replicates are recommended for each sample.
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).
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) of circumvallate papilla. Scale bars: 1 mm in A1, A3, A4, B1, B2; 200 µm in A2; 100 µm in B3, B4, B5, and B6. Please click here to view a larger version of this figure.
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 µm for B1,B2, B3, and B4; 25 µm for high power images in top right corner. Please click here to view a larger version of this figure.
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 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 (>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/µL (around 200 cells per square in hemocytometer). The optimal concentrations range from 500 to 2500 cells/µL.
The authors have nothing to disclose.
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.
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 |