Generation and Culture of Lingual Organoids Derived from Adult Mouse Taste Stem Cells

The protocol presents a method for culturing and processing lingual organoids derived from taste stem cells isolated from the posterior taste papilla of adult mice.

Organoids are powerful in vitro technology used for drug screening and understanding biological processes. So, generating organoids that model the tongue is crucial for studying taste cell development and regeneration. Traditional in vivo taste studies can be expensive and time-consuming.

This organoid protocol offers a standardized, reproducible alternative that minimizes those challenges while allowing for higher throughput experiments. After euthanizing an adult mouse, use large sterile dissection scissors to cut the cheeks and break the jaw. Then, lift the tongue and cut the lingual frenulum to separate the tongue from the floor of the oral cavity.

Cut the tongue out and collect it in sterile ice-cold dPBS with calcium and magnesium. Under a dissecting microscope, remove and discard the anterior tongue by cutting just anterior of the inter malar eminence with a razor blade. Then using a delicate task wipe to remove any hair and excess liquid from the posterior tongue.

Next, fill a one milliliter syringe with 200 to 300 microliters of injection enzyme solution and insert a 30 gauge half-inch needle just above the inter malar eminence until just anterior of the circumvallate papillae, or CVP. Inject the enzyme solution underneath and at the lateral edges of the CVP between the epithelium and the underlying tissues. Withdraw the syringe slowly and continuously from the tongue as the solution is being injected.

Incubate the tongue and sterile calcium magnesium-free dPBS at room temperature for precisely 33 minutes. Using extra-fine dissection scissors, make small cuts in the epithelium bilaterally and just anterior of the CVP. Then gently peel the epithelium by lifting it with fine forceps.

Once the trench epithelium is free of the underlying connective tissue, place it into two milliliter microcentrifuge tube, pre-coated with FBS. Add the dissociation enzyme cocktail to the tubes containing the peeled CVP epithelia and incubate them in a 37 degree Celsius water bath for 45 minutes with brief vortexing every 15 minutes. During the last 15 minutes of incubation, prewarm 0.25%Trypsin-EDTA in the water bath.

Following incubation, vortex the tubes in triturate with a glass Pasteur pipette for one minute. After tissue pieces settle, pipette the supernatant containing the first collection of dissociated cells into new FBS coated 1.5 milliliter microcentrifuge tubes. Spin the supernatant for five minutes at 370 times G and four degrees Celsius to pellet the cells.

Remove the resulting supernatant, then resuspend the cell pellet in FACS buffer and keep it on ice. To dissociate the remaining tissue pieces in the original two milliliter microcentrifuge tubes, add pre-warmed 0.25%Trypsin-EDTA and incubate at 37 degrees Celsius for 30 minutes with brief vortexing every 10 minutes. Then, triturate the tissue pieces with a glass Pasteur pipette for one minute.

After the tissue pieces settle, pipette the supernatant into the 1.5 milliliter microcentrifuge tubes containing the previously collected dissociated cells in FACS buffer. Spin the tubes with the dissociated cells. After removing the supernatant, resuspend the cell pellets in FACS buffer and keep them on ice.

Then, isolate the Lgr5-GFP positive cells via FACS using the green fluorescent protein channel as described in the text manuscript. Transfer the desired number of Lgr5 positive suspended cells into a new microcentrifuge tube. Spin the tube for five minutes at 370 times G and four degrees Celsius to pellet the cells.

Remove the supernatant and place the tube on ice. Gently resuspend the cell pellet in the appropriate amount of matrix gel with gentle pipetting. Then keep the microcentrifuge tube on ice in a 50 milliliter conical tube to prevent the matrix gel from gelling.

Add 15 microliters of the matrix gel in cell mixture in the center of each well of a 48-well plate. To ensure an even distribution of cells, mix the matrix gel and cell mixture by pipetting up and down after plating every three wells. Place the plate in an incubator at 37 degrees Celsius, 5%carbon dioxide and 95%humidity for 10 minutes to allow gelling of the matrix gel.

Then, add 300 microliters of room temperature WENRAS media supplemented with the rock inhibitor, Y27632, to each well and return the plate to the incubator. Two days after plating, remove the media from each well using a one milliliter pipette or via vacuum aspiration, ensuring no cross-contamination between conditions. Add 300 microliters of WENRAS media down the side of the well, taking care not to disrupt the matrix gel and return the plate to the incubator.

When lingual organoids are cultured using WENR media, they do not grow efficiently. However, after adding A 83-01, a TGF-beta signaling inhibitor, and SB202190, P-38 map kinase signaling inhibitor, robust growth is observed. Interestingly, removing these inhibitors from the media after six days results in higher expression of general taste receptor cell marker, Kcnq1, suggesting that A 83-01 and SB-202190 hinder taste cell differentiation.

Thus, optimal growth and differentiation are obtained by culturing organoids in WENRAS media from day zero to six and WENR media from day six to 12. Mature organoids contain both taste cells marked by keratin-8 and non-taste epithelial cells marked by keratin-13. Further, keratin-13 is expressed at higher levels than all three taste receptor cell markers, suggesting that organoids are predominantly composed of non-taste epithelial cells.

The organoids express all taste receptor cell types. Type one cells marked by Entpd2 and bitter type two cells marked by Gnat3 are highly expressed in taste organoids, while sour sensing type three cells marked by Car4 are less common. The taste receptor cells are randomly distributed in the organoids rather than in discrete taste bud structures observed in vivo.

Make sure to include some tissue posterior to the CVP when dissecting the tongue from the oral cavity. Additionally, make sure both trenches pop out and are obtained when peeling the epithelium.