To begin, rinse extracted tooth with a sterile saline solution to remove any blood. Carefully cut the tooth longitudinally into two pieces using a carbide fissure burr while irrigating with sterile saline. Afterward, transport the tooth and its intact pulp in sterile alpha-MEM media on ice to the cell and tissue culture laboratory.
Then place the extracted tooth sample in sterile culture Petri dish and thoroughly rinse three to four times with sterile PBS. Using a sharp excavator and forceps, carefully remove the cavity pulp tissue and clean a thoroughly with sterile PBS to remove blood traces. Now divide the dish surface into four parts by putting 1 to 2 milliliters of sterile PBS in each part.
Place the pulp tissue sample in one area and cut it into small pieces using a surgical blade. Transfer the tissue pieces by lifting them into successive areas with PBS. Meanwhile, add approximately 0.8 to 1 milliliter of alpha-MEM containing non-essential amino acids with 10%FBS and an antibiotic cocktail in a six-well plate.
And move it to spread the media uniformly. Various stages of primary culture development of dental pulp stem cells are shown. Rounded bubble type cells from dental tissue were observed on the second day of seeding.
A mess-like network of cells coming out of tissue was observed on the fourth day. However, by the 13th day, the number of cells that emerged out of the explant increased. And by the end of the second week, most of the explants got surrounded by many adherent cells with spindle shape morphology.
