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Objectives: This study aimed to simplify the collection, isolation and cryopreservation procedure of human dental pulp stem cells (DPSCs) to ease the establishment of dental stem cell banking., Design: Extracted third molars were collected and stored either in growth medium or in gentamicin-saline (480μg/ml) for 6, 9 or 12h. DPSCs were isolated and subjected to cryopreservation by a controlled-rate or rapid freezing method in 5 or 10% DMSO. Flow cytometry and growth pattern of DPSCs before and after cryopreservation were conducted., Results: Rate of contamination by which the extracted teeth were stored in control and gentamicin-saline were 9.1% (N=33) and 2.3% (N=43), respectively. Successful cell isolation rate of teeth preserved in gentamicin-saline at 6h (92.9%) was comparable to those of growth media group (90.3%). At 9 and 12h, the rates dropped significantly to 75% and 54%, respectively. Cryopreservation by controlled-rate freezing either in 5 or 10% DMSO resulted in a significantly higher percentage of viable cells than by rapid freezing. Cells conserved by controlled-rate freezing in 5% DMSO showed a pattern of growth similar to control unfrozen cells; 10% DMSO significantly deteriorated the growth pattern of the cells. After thawing, DPSCs conserved by controlled-rate freezing still expressed stemness characteristics, although hematopoietic stem cell markers were slightly increased., Conclusion: Gentamicin-saline was effective in preserving human teeth for DPSC isolation. Controlled-rate freezing in 5% DMSO gave the highest rate of cell viability. This study simplifies the storage conditions and proposes a simple method for cryopreservation of DPSCs., (Copyright © 2017 Elsevier Ltd. All rights reserved.)