Establishing a Co-Culture of Dental Pulp Cells and Trigeminal Neurons for Cross-Communication

Begin with a mesenchymal stem cell-rich suspension obtained from mouse dental pulp tissue.

Incubate to allow cell adhesion and proliferation, forming a monolayer.

Place a porous membrane insert coated with laminin into the well containing the dental pulp cells.

Seed the insert with a co-culture medium containing trigeminal neurons derived from the mouse brain. Incubate to allow neuron attachment.

Replace the medium with a co-culture medium containing growth regulators such as uridine and 5-fluoro-2 deoxyuridine.

These molecules enter the cells and control the growth of actively proliferating mesenchymal stem cells by inhibiting DNA synthesis.

Over time, the mesenchymal cells secrete small signaling molecules that pass through the porous membrane to stimulate the trigeminal neurons.

This stimulation causes the neuronal processes of the trigeminal neurons to elongate toward the mesenchymal cells. Additionally, the neurons secrete signaling molecules, enabling cross-communication.