Evaluation of two human dental pulp stem cell cryopreservation methods.

Dental pulp is a promising source of mesenchymal stem cells for use in cell therapy and regenerative medicine. Methods for storing stem cells with minimum compromise of cell viability, differentiation capacity and function should be developed for clinical and research applications. The aim of this study was to evaluate whether human dental pulp stem cells (hDPSCs) isolated and cryopreserved for 1, 7 and 30 days maintain viability and expression of specific stem cell markers. Human dental pulp stem cells were isolated from 23 healthy patients aged 18 to 31 years. Dental pulp was enzymatically dissociated, and CD105+ cells were separated using the Miltenyi™ system. The hDPSCs were cryopreserved using the Kamath and Papaccio methods. Post-cryopreservation viability was measured by flow cytometry (7AAD) and by the expression of the phenotype markers CD105+/ CD73+, CD34-/CD45-. The Papaccio method showed greater cell viability for cells that had been frozen for 30 days (59.5%) than the Kamath method (56.2%), while the Kamath method provided better results for 1 day (65.5%) and 7 days (56%). Post-cryopreservation expression of the markers CD105+/CD34- was greater after 1 and 7 days with the Kamath method and CD105+/CD45- were expressed after all 3 cryopreservation times. There was greater expression of CD73+ in the hDPSCs after 1 and 7 days with the Kamath method, and after 30 days with the Papaccio method. These results suggest that hDPSCs express mesenchymal stem cell markers after cryopreservation. However, cryopreservation time may affect marker expression, probably by altering the spatialconfiguration of cell membrane proteins or by compromising cells at a certain level of differentiation.