This protocol demonstrate an enhanced method for procuring human dental epithelial cells. It is difficult to identify the aggregated cell pellet during the procedures. Be careful when you handle supernatant to prevent the loss cell populations.
To begin, harvest dental follicles during the surgical extraction of impacted third molars. Start cell isolation procedures or store the dental follicles at four degrees Celsius in DPBS with 3%penicillin-streptomycin. To wash the dental follicles, hold the dental follicle with tweezers and place it in washing tube 1.
Gently shake it 10 to 15 times. Take it out and place it in tube 2. Again, shake it 10 to 15 times.
Finally, take it out and place it in tube 3, then shake it. After washing three times, place the dental follicle in a 60 millimeter culture dish. Mince the tissue with tissue scissors until the dental follicle has a pulpy or squishy appearance.
Use a small side section of the culture dish to minimize tissue loss. Mix one milliliter each of collagenase type 1 and protease solution in a 15 milliliter conical tube. Transfer the minced tissue into the 15 milliliter conical tube, then gently shake it and incubate it for one hour at 37 degrees Celsius.
Add five milliliters of 0.05%trypsin-EDTA to the tube. Shake it and incubate for 15 minutes at 37 degrees Celsius. Prepare keratinocyte medium containing keratinocyte serum-free medium and 10%fetal bovine serum.
Add three milliliters of keratinocyte medium into a new 50 milliliter conical tube. Place a 40 micrometer strainer on top of this tube. Next, aspirate the supernatant from the tube containing the tissue with a 10 milliliter serological pipette and pass it through the strainer.
Transfer the collected suspension to a 15 milliliter conical tube. Centrifuge the tube and remove the supernatant. Then add three milliliters of keratinocyte serum-free medium.
Centrifuge for another three minutes and remove the supernatant. Plate the single cell population into a 60 millimeter culture dish using the keratinocyte serum-free medium. Maintain the culture dish with a final volume of five milliliters in a humidified atmosphere of 5%carbon dioxide at 37 degrees Celsius.
Cells with epithelial morphology appeared within seven to 10 days after plating single cell populations. The number of cobblestone-shaped epithelial cells ranged from one to 10 at the time of emergence and the cells expanded into colonies over time. The most crucial step is mincing the dental follicle into small particles.
It enhances the separation of single cells from follicle tissue. Mesenchymal cells can be isolated from human dental follicle. Serum containing culture environment selects mesenchymal cells and inhibits epithelial growth from single cell populations.
This protocol provide the method of procuring the human dental epithelial cells. Dental follicle-derived epithelial cells can be utilized for the study of dental epithelial-mesenchymal interactions.
