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