Isolation, Culture, and Characterization of Dental Pulp Stem Cells from Human Deciduous and Permanent Teeth

Begin by securing the extracted human tooth in place with dental forceps. Then cut the tooth using a diamond disc connected to a dental handpiece and a water coolant. Using a dental excavator, remove the pulp tissue from the tooth.

Next, use a sterile surgical blade to carefully mince the pulp tissue into small fragments. Then place these fragments into a mini tissue grinder with PBS to form a homogenous mixture. Transfer the minced tissue fragments into a 15 milliliter tube and digest them using a mixture of collagenase type one and dispase.

Incubate the tissue in the enzyme mixture at 37 degrees Celsius for two hours. After incubation, neutralize the enzymes. Next, transfer the digested tissue to a 15 milliliter tube and spin at 300 G for five minutes at room temperature.

Then carefully discard the supernatant and re-suspend the pellet in fresh DMEM, supplemented with 10 to 20%FBS. For cell culture, carefully transfer the cell suspension to a 25 square centimeter culture flask. Incubate the cells at 37 degrees Celsius in 5%carbon dioxide.

After two to three days, using a sterile serological pipette, carefully aspirate the medium and replace it with a fresh medium. Next, for the characterization of DPSCs, turn on the flow cytometer and confirm the mesenchymal stem cell nature of the isolated cells. The successful differentiation of DPSCs into osteoblasts, adipocytes, and chondrocytes validates the multi-potent nature of DPSCs.

The doubling time of DPSCs was consistent with that reported for mesenchymal stem cells, suggesting a healthy and proliferative cell population. The flow cytometry analysis showed that a majority of the DPSCs were positive for CD-90, CD-73, and CD-105, confirming their identity as mesenchymal stem cells. Moreover, less than 2%of the cells were positive for CD-34, CD-45, and HLADR, confirming the absence of significant hematopoietic or endothelial cell contamination.