Dental pulp stem cells (DPSCs) are mesenchymal stem cells with high self-renewal potential that have the ability to differentiate into several cell types. Thus, DPSCs have become a promising source of cells for several applications in regenerative medicine, tissue engineering, and stem cell therapy. Numerous methods have been reported for the isolation, expansion, and preservation of DPSCs. However, methods are diverse and do not follow specific rules or parameters, which can affect stem cell properties, adding more variation to experimental results. In this review, we compare and analyze current experimental evidence to propose some factors that can be useful to establish better methods or improved protocols to prolong the quality of DPSCs. In addition, we highlight other factors related to biological aspects of dental tissue source (e.g., age, genetic background) that should be considered before tooth selection. Although current methods have reached significant advances, optimization is still required to improve culture stability and its maintenance for an extended period without losing stem cell properties. In addition, there is still much that needs to be done toward clinical application due to the fact that most of DPSCs procedures are not currently following good manufacturing practices. The establishment of optimized general or tailored protocols will allow obtaining well-defined DPSCs cultures with specific properties, which enable more reproducible results that will be the basis to develop effective and safe therapies.