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

High yield and quality of cells from complex tissues are essential to commonly used experimental analysis, such as single cell RNA sequencing and primary stem cell cultures. This protocol generates healthy cells at high yield and quality from different regions and tissue compartments of the mammalian tongue, including tongue epithelium and connective tissue. To separate the epithelium from the mesenchyme of an E 12.5 mouse tongue, transfer the euthanized pregnant female mouse to the surgical area and wet the mouse abdomen with 70%ethanol to prevent fur from getting into the operating site.

Open the abdomen using dissecting scissors to expose the uterine horns carrying the embryos, dissect the uterine horns, and transfer them to a 15 milliliters of fresh Tyrode’s solution in a 100 millimeter culture dish. Under a dissecting microscope, use mini scissors and fine forceps to dissect the embryos from the uterine horns. Open the mouth cavity with the fine forceps and dissect the tongue from the mandible using mini scissors.

Wash the tongues with 15 milliliters of fresh sterile Tyrode’s solution in a 100 millimeter culture dish. Then transfer the tissues to two milliliters of an enzyme mixture of dispase and collagenous in a 35 millimeter culture dish and incubate for 20 minutes at 37 degrees Celsius. Transfer the tongues to 15 milliliters of fresh sterile Tyrode’s solution and gently separate the mesenchyme and the epithelium from the ventral side using fine forceps.

Wash the separated epithelia and mesenchyme twice in 15 milliliters of fresh sterile Tyrode’s solution. To separate the tongue epithelium from the underlying connective tissue of an adult mouse, transfer the euthanized mouse to the surgical area and wet the mouse head using 70%ethanol to prevent fur from getting into the oral cavity. Use dissecting scissors to cut the corners of the mouth along the cheek to open the oral cavity.

Dissect the tongue with a mandible, wash it and place it in a plastic dish with a layer of plastic wrap. Using surgical forceps to hold the tongue, inject the enzyme mixture of dispase and collagenase in the subepithelial space of the tongue through the cutting edge of the posterior tongue. Inject one milliliter of enzyme mixture evenly to the whole tongue for tissue collection, then inject 0.5 milliliters of enzyme mixture locally to the anterior tongue for tissue collection from the tongue tip or to the posterior tongue for circumvallate papilla tissue collection.

Wrap the tongue with plastic wrap and incubate it for 30 minutes at 37 degrees Celsius. Use mini scissors to dissect the circumvallate papilla and tongue tip. Then transfer a tissue to 15 milliliters of fresh sterile Tyrode’s solution.

Separate the epithelium from the underlying connective tissue in the enzyme digested subepithelial space using mini scissors and trim the tissues to a proper size, according to the requirement of downstream experiments. Wash the separated epithelium and underlying connective tissue twice in 15 milliliters of fresh sterile Tyrode’s solution in a 100 millimeter culture dish. Transfer the tissues to three milliliters of 0.25%trypsin EDTA in a new 35 millimeter culture dish.

Incubate the dish for 30 minutes at 37 degrees Celsius, gently agitating the tissues every five minutes with one milliliter pipette tips. Add 500 microliters of 5%FBS in DMEM F12 to stop the reaction and transfer the medium to a five milliliter low binding centrifuge tube. Centrifuge the cell suspension at 200 times G for eight minutes at room temperature and remove the supernatant.

Gently resuspend the cells in three milliliters of DMEM F12 containing 10%FBS and 1%BSA, and filter the cells with a 70 micrometer cell strainer, followed by a 35 micrometer cell strainer. Centrifuge the cell suspension at 200 times G for eight minutes at room temperature, then remove most of the medium, leaving 50 to 300 microliters as the final volume to resuspend the cells. In the embryonic mouse tongue, a gap in the subepithelial space is visible after proper enzyme digestion.

In the adult mouse tongue, a successful enzyme injection is indicated by swelling in the injected areas, which suggests that the enzyme can be held by the tongue. After pooling E12.5 tongues’epithelial sheets and thin layers of mesenchyme, manual cell counting demonstrated that the protocol yielded 63, 917 cells in total with a viability of 95.2%from the epithelial sheets, and 294, 333 cells in total with a viability of 96.3%from the mesenchyme. When 10 adult tongues at eight weeks of age were used, the protocol yielded 187, 333 cells with a viability of 95.4%from epithelial sheets of the tongue tip, 544, 000 cells with a viability of 96.3%from epithelial sheets of circumvallate papillae, and 150, 500 cells with a viability of 93%from connective tissues.

Following this protocol, the dissociated cells can be used for single cell RNA sequencing and 3D test organoid culture to define unrecognized test progenitor cells under the lingual epithelium.