Tooth Storage, Dental Pulp Stem Cell Isolation, and Clinical Scale Expansion without Animal Serum

Introduction: Dental pulp stem cells (DPSCs) have therapeutic potential for dentin and dental pulp regeneration. For regenerative approaches to gain clinical acceptance, protocols are needed to determine feasible ways to store teeth, isolate DPSCs, and expand them to clinical scale numbers. Methods: In this study, 32 third molars were obtained from patients and immediately placed in saline or tissue culture medium followed by overnight storage at 4 C or immediate isolation of DPSCs. Upon isolation, cells were expanded in medium containing either fetal bovine serum (FBS) or human serum (HS). Cell proliferation (population doubling time [PDT], cell surface marker expression, and multi-potency were compared between DPSCs in FBS and DPSCs in HS. Results: The time frame of storage and storage medium did not affect the ability to isolate DPSCs. However, using HS instead of FBS in the initial isolation of DPSCs significantly decreased (P < .01) the isolation success rate from 89% (FBS) to 23% (HS). Yet, incorporating fibronectin in the DPSC initial isolation (using HS) significantly (P <. 01) increased the isolation success rate to 83%. Interestingly, it was found that the proliferation rate was significantly (P < .05) higher for DPSCs in HS (PDT = 1.59 +/- 0.46) than that for DPSCs in FBS (PDT = 2.84 +/- 2.5). Finally, there was no difference in the expression of CD73, CD90, CD105, or multipotency (as measured by osteogenic, adipogenic, and chondrogenic differentiation) between DPSCs in FBS and DPSCs in HS. Conclusions: These findings show a clinically feasible method of storing third molars for the isolation of DPSCs. Additionally, DPSCs can be isolated and expanded to clinical scale numbers in media devoid of FBS and still maintain their phenotypic properties.