The dental pulp samples were collected from the intact 3rd molar extraction of 8 patients, ages 16 -25 years. Facilitated by Dra. Belinda I. Beltran- Salinas from Faculty of Dentistry of the Universidad Autónoma de Nuevo Leon / Ministry of Health, they were all recollected the morning they were to be processed.After extraction of the dental pulps, the procedure followed is as follows. Add 4.75 mL of PBs in a GentleMACS C tube and insert it in the GentleMACS instrument ensuring the sample is in touch with the tube vanes. Run either program A or B. Remove the tube and digest the pulps in a 3mg/mL collagenase I and 4mg/mL dispase solution. Incubate for 1 hr at 37°C in a water bath. Once incubated, reintroduce the tube on the GentleMACS and run programs B or C.
After tissue was disgregated, stem cells were isolated from all other tissue cells using the magnetic separation technique (MiltenyiBiotec), in which CD271 and CD44 monoclonal antibodies were used.
The cells were observed in an inverted microscope finding elongated, flattened, and fibroblastic dental pulp stem-cell morphologies. Subsequently, by means of flow cytometry, these cells were quantified and presented positive results for both markers, CD271 (0.6) and CD44(95.7 + -3.8) in 10,000 cells.
The Magnetic Activated Cell Sorting (MACS) technology It has proved effective in the identification of CD 271 and CD44 cells demonstrating their usefulness in processing samples of dental pulp comparing it with traditional methods currently used enzymatic
Keywords: Cell culture, Methodology, Pulp, Technology and Teeth
See more of: Pulp Biology & Regeneration Research