Use of Trowell-Type Organ Culture to Study Regulation of Dental Stem Cells

The Trowell-type organ culture is used to culture tissue explants. Here, we show how to use this method to study the stem cells in continuously-growing teeth. With this technique, we can study the stem cells and their behavior in the niche and screen the effect of various molecules on the survival and maintenance of stem cells.

This method can be applied to study the stem cells of other organs as well, such as skin or mammary gland. Begin by placing 30-millimeter metal grids with one to two-millimeter diameter holes in a 35-millimeter Petri dish. Fill the place with media until the media reaches the grid surface.

Then, incubate the plate at 37 degrees Celsius until the tissue is isolated and ready to culture. After decapitating the mouse, remove the skin to expose the mandible and cut through the masseter muscles to separate the mandible from the maxilla and the rest of the head. Once the mandible is isolated, remove the tongue and maximum soft tissue.

Split the mandible at the midline by cutting through the symphysis. Place the mandible in the Petri dish containing PBS on the ice. Use a disposable 20 by 26 gauge hypodermic needle to dissect the incisor.

Then, clean the soft tissue and muscle tissue from the bone surface for better visualization. For mandibles obtained from animals younger than 10 days, use disposable 20 by 26 gauge hypodermic needles to open each half of the mandible longitudinally to expose the incisor tooth. Gently detach the incisor from the surrounding bone.

Cut the proximal end that contains the cervical loop and remove the mineralized enamel and dentin matrix. Keep the collected proximal ends in Dulbecco's PBS on ice until ready to culture. For mice older than 10 days, open each half of the mandible longitudinally to expose the incisor tooth.

Use tweezers to grip the mandible and break the bone to expose the tooth. Cut the proximal end that contains the cervical loop. Keep the collected proximal ends in Dulbecco's PBS on ice until ready to culture.

Carefully place the rectangle filters on top of the grid in a pre-warmed culture dish and properly orient the tissue pieces. Incubate the plate at 37 degrees Celsius, 5%carbon dioxide, and 90 to 95%humidity. Carefully change the medium on alternate days, avoiding the formation of air bubbles.

Monitor the tissue growth and capture images daily. Remove the culture medium from the dish and carefully add ice-cold methanol to the tissue. Then, incubate it for five minutes.

Transfer a filter carrying the tissue explant to the sampling tube filled with fixing solution. Fix the explant in 4%paraformaldehyde prepared in PBS for maximum overnight at four degrees Celsius. The epithelial stem cells reside in a niche called the cervical loop, located at the proximal end of the incisor.

The cervical loop comprises an inner and outer enamel epithelium that encases the stellate reticulum, a core of loosely-arranged epithelial cells. Each incisor has one labial and one lingual cervical loop but only the labial cervical loop contains the stem cells. The proximal end of an incisor, isolated from two-day postnatal Sox2-GFP mice, were captured, enabling the identification and visualization of the Sox2-expressing incisor epithelial stem cells.

In cervical loops isolated from the Sox2-GFP and Fucci red transgenic mice, GFP-positive stem cells and Fucci-red-positive non-proliferative cells were identified. The Sox2-GFP expression was used as a reporter to analyze the effect of Wnt/beta-catenin signaling activator BIO, which negatively affected Sox2-expressing stem cells as well as the GFP expression. This technique can be optimized for live imaging of the stem cell behavior.

Pressed or cultured explants can also be processed enzymatically to single-cell suspensions for further analysis.