Analyzing Tooth Germ and Trigeminal Ganglia Interactions Using a Microfluidic Co-culture System

Take a microfluidic device coated with a synthetic polymer and an adhesion protein.

The device consists of wells and interconnected culture chambers separated by microgrooves.

The chambers contain punched holes for tissue placement.

Obtain the trigeminal ganglia, a collection of trigeminal neuron cell bodies, and tooth germs, prenatal structures that develop into adult teeth, from a mouse embryo.

Place the tissues in the holes and add the respective culture media.

Nutrients and vitamins in the tooth germ media facilitate tooth germ survival during culturing.

The coated device surface and growth factors in the trigeminal ganglia media facilitate neurite and axonal outgrowth.

The axons reach the tooth germ chamber through the microgrooves.

The tooth-derived signals lead to neuronal repulsion, allowing the axons to surround the tooth germ without innervating it.

Wash the culture chambers with buffer and fix the tissues with a fixative.

The co-culture is now ready for further analysis.

After dissection of the trigeminal ganglia and tooth germs, remove the laminin from the microfluidic devices, and fill the chambers with 200 microliters of the respective media. With forceps, gently transfer the dissected trigeminal ganglia and tooth germs into the holes created with a biopsy punch. Make sure that the tooth germs do not float and that they sink until they contact the coverslips.

Culture the samples in an incubator at 37 degrees Celsius and 5% carbon dioxide. Change the culture medium every 48 hours for 10 days. To change the medium, first, remove the medium by pointing the pipette towards the external side of the wells. Then, add the fresh pre-warmed medium on the side of the wells located opposite to the chambers.

During the culture period, image the co-cultures at any time using light microscopy. After the culture period, wash the chambers by pipetting 150 microliters of PBS into one well per chamber, and letting PBS flow through the chambers. Repeat the wash a total of three times.

Following the last wash, remove PBS and fix the samples by pipetting 150 microliters of 4% paraformaldehyde in PBS into one well per chamber and allowing it to flow through to the other chamber. Incubate the device at room temperature for 15 minutes. Then, wash the chambers twice with PBS and proceed with further analysis, such as immunofluorescence staining and imaging of the outgrowth.