Advances in dental pulp stem cell (DPSC) biology and behaviour have promised much in the field of regenerative medicine. Their recent use in clinical trials for bone repair enforces the notion that DPSCs can be used successfully in patients; however they display diverse characteristics under different culture conditions. Since the success of any stem cell culture is regulated by its own micro-environment, it is imperative to optimise the growth conditions and establish a generic protocol for maintenance and scale-up. This study focused on optimisation of long-term culture conditions of human exfoliated deciduous teeth (SHED) in comparison with DPSCs, employing three commonly used basal media – knockout Dulbecco's modified Eagle's medium (KO-DMEM), α-MEM and DMEM/F12. Based on their characterisation with respect to morphology, growth kinetics, cell surface marker expression, differentiation capacity and plating density, our findings suggest that cells can be expanded with the highest efficiency in KO-DMEM medium supplemented with 10% FBS. Additionally, under our standardised xeno-free (10% human plasma) growth conditions, DPSCs displayed and retained their multipotent attributes until late passages. The differences in the growth and differentiation characteristics between SHED and DPSCs are shown, and certify SHED can be a key element in tissue engineering.