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

This protocol involves the use of animals and all the procedures were approved by Ethical Committees on the Use and Care of Animals and the Animal Facility at the University of Helsinki.

1. Preparation of the organ culture dish

1. Perform all procedures in a laminar flow hood. Clean work surfaces with 70% ethanol and use autoclaved glass instruments and solutions. Sterilize scissors and other metal equipment in a glass-bead sterilizer.
2. Prepare filters normally stored in 70% ethanol by washing them three times in 1x PBS to remove ethanol (Figure 1). Cut filters into rectangular pieces (3 x 3-5 x 5 mm).
   NOTE: Washed filter pieces can be stored for several days in 1x PBS at 4 °C.
3. Prepare the culture medium (1:1 DMEM:F12 supplemented with 1% [v/v] 200 mM L-alanyl-L-glutamine dipeptide in 0.85% NaCl, 10% [v/v] FBS, 150 µg/mL ascorbic acid, and 0.2% [v/v] penicillin [10,000 I.U./mL] and streptomycin [10,000 µg/mL]). Store at 4 °C.
4. Place the 30 mm metal grids (with 1-2 mm diameter holes that enable tissue imaging) in a 35 mm Petri dish. Add sufficient media to reach the grid surface without producing air bubbles. Pre-warm the prepared culture dish at 37 °C until the tissue is isolated and ready for culture (in a standard incubator with 5% CO₂ and 90%-95% humidity).

2. Incisor dissection and isolation of the proximal end

1. Sacrifice the animals following an approved animal care protocol.
2. Decapitate the mouse and dissect the mandible. To do so, first remove the skin to expose the mandible and cut through the masseter muscles to separate it from the maxilla and the rest of the head.
3. Once the mandible is isolated, remove the tongue and as much soft tissue as possible.
4. Collect all the mandibles and keep them in a Petri dish containing PBS on ice, as this enhances the viability of the tissues.
5. Transfer one mandible to a glass Petri dish and use disposable 20/26 G hypodermic needles to dissect the incisor under a stereomicroscope. Split the mandible at the midline, cutting through the symphysis. Clean the soft and muscle tissue away from the bone surface for better visualization.
   ​NOTE: A glass Petri dish is essential, as this will not blunt the needles.
6. Mandibles obtained from animals younger than 10 days are softer and more fragile, therefore, use disposable 20/26 G hypodermic needles to open each half of the mandible longitudinally to expose the incisor tooth. For mice older than 10 days, use tweezers to grip the mandible and break the bone to expose the tooth.
7. Gently detach the incisor from the surrounding bone and cut off the proximal end, which contains the cervical loop.
8. Cut the proximal end and remove the mineralized enamel and dentin matrix.
9. Keep the collected proximal ends in Dulbecco's PBS on ice until ready for culture.

3. Culture

1. Carefully place a filter rectangle on the top of a grid in a pre-warmed culture dish.
2. Use a stereomicroscope to properly orient the tissue pieces.
3. Place in a standard incubator at 37 °C with 5% CO₂ and 90%-95% humidity.
4. Change the medium every other day and replace with fresh medium, carefully avoiding formation of air bubbles. Monitor tissue growth and photograph daily using a camera attached to the stereomicroscope.

4. Adding soluble factors to the culture

1. Supplement the culture medium with soluble factors and molecules of interest to study their effect on regulation of SCs.
   ​NOTE: The administration protocol for any molecule (growth factors, signaling molecules, blocking antibodies, pharmacological compounds such as inhibitors or activators, vectors, etc.) depends on its half-life and solubility. These parameters also determine the appropriate control to be used.

5. Molecular and histological analyses

1. Remove the culture medium.
2. Carefully add ice-cold methanol to the tissues to prevent detachment from the filters.
3. Leave methanol for 5 min.
4. Transfer filters carrying tissue explants to sampling tubes.
5. Fix the explants in 4% paraformaldehyde in PBS for 10-24 h at 4 °C.
6. Proceed with established protocols for histological processing (paraffin, frozen, etc.) or immunostainings.

6. Culture of tissue slices

NOTE: There are several variations to this protocol, which are all equally successful and are a matter of personal choice, depending on the speed and the skill of the user. These refer to the buffer used to collect and maintain tissue viability. For this purpose, Krebs buffer, PBS supplemented with 2% glucose and antibiotics, or PBS can be used. If Krebs buffer is used, it should be made 1 day in advance and kept at 4 °C.

1. Before the experiment, prepare 4%-5% low-melting point agarose by dissolving 2-2.5 g of low-melting point agarose in 50 mL of boiling 2% glucose/PBS, after which the solution should be placed in a 45 °C water bath.
2. Set up the vibratome, wash with 70% ethanol, and fill with ice-cold 2% glucose/PBS supplemented with antibiotics (100 U/mL penicillin, 100 mg/mL streptomycin).
3. Dissect the proximal ends of the incisor and collect them in the ice-cold 2% glucose/PBS supplemented with antibiotics.
4. Place one proximal end of the incisor in the mold containing 4%-5% low-melting point agarose. Under a stereomicroscope, orient the piece in the desired direction and leave on ice for the agarose to harden.
5. Trim the hardened agarose block and place it in the vibratome. Cut thick slices (150-300 µm).
6. Collect the slices in a Petri dish containing ice-cold 2% glucose/PBS supplemented with antibiotics.
7. Use a spatula to transfer the thick slices on a filter rectangle placed on the pre-warmed grid prepared as in section 1.3.
8. Incubate the thick slices in the incubator and proceed with imaging (Figure 2).