Stem cells isolated from dental pulp possess the capacity for self-renewal and the potential for multi-lineage differentiation. However, dental pulp stem cells have different characteristics in terms of their culture conditions. The success of stem cells culture is governed by its micro-environmental niche. Therefore, we studied the effects of culture niche on long-term expansion of dental pulp stem cells in terms of cell morphology, growth kinetics, senescence pattern, cell surface marker expression differentiation capacity, and seeding plating density of dental pulp stem cells in four different, widely used media composition Among the various basal media tested, α-minimum essential media and knock out-minimum essential media supplemented with 10% fetal bovine serum were found to be the most optimal media composition in preserving the phenotypic characteristics and differentiation potential for prolonged periods as compared with DMEM-F12 and DMEM-LG. Plating density has been shown to affect overall yield. As a conclusion, the adoption of an appropriate culture system significantly improved cell yield, thus enabling the attainment of sufficient yields for therapeutic applications economizing in terms of cost of production and minimizing seeding cell density for maximum yield.