Primary Culture of Dental Pulp Stem Cells

We are investigating the potential of DPCs in cell-based and cell-free applications for disease models, while studying the cellular and molecular mechanisms involved in tissue regeneration. Additionally, we are exploring the role of stem cell derived security factors in controlling stem cell fate and promoting tissue regeneration. Our current focus is on using 3D culture techniques to gain deeper understanding of molecular mechanisms, genetic and epigenetic changes associated with tissue regeneration.

We are incorporating advanced techniques such as next-gen sequencing, RNA sequencing and chip sequencing to unravel regulatory domains governing various cellular processes. Using explant methods to isolate DPC, a homogeneous stem cell population can be efficiently harvested, free from other cells like endothelial and pericytes. These cell types remain in the cell culture when DPCs are established using enzymatic procedures.

Our research using DPCs as a cellular model for drug staining and tissue regeneration can develop novel therapies, advance personalized medicine, and have far reaching implications in the regenerative medicine, including craniofacial reconstruction and invaluable bone regeneration after dental extraction. We are currently investigating how DPCs can be used to address bone defects, such as long bone and calvary defects. Additionally, we are exploring the potential therapeutic implications of secreted molecules derived from DPCs in various disease models, including neuroregeneration and glucagon.

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 it thoroughly with sterile PBS to remove blood traces. Now divide the dish surface into four parts by putting one to two 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 one 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 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 explants increased, and by the end of the second week, most of the explants got surrounded by many adherent cells with spindle-shaped morphology.