BACKGROUND: Dental pulp-derived mesenchymal stromal cells (DPMSCs) represent a promising avenue for regenerative medicine. However, the therapeutic potency of DPMSCs is substantially influenced by their functional heterogeneity emerging during ex vivo expansion. Therefore, identifying and selecting the most potent subpopulation of DPMSCs following expansion is key to improving therapeutic efficacy and consistency. Nevertheless, conventional methods for isolating stem cell subpopulations are largely impractical for clinical-scale bioprocessing, primarily due to their high cost and limited throughput. Recent research has unveiled a strong correlation between the biophysical characteristics of culture-expanded stem cells and their functional attributes, thereby raising the prospect of employing high-throughput, cost-effective biophysical sorting techniques to isolate functionally distinct subpopulations. METHODS: A high-throughput microfluidic chip was implemented to perform label-free separation of culture-expanded DPMSC subpopulations based on their varying cell sizes, utilizing the principle of Dean flow fractionation. RESULTS: Leveraging this microfluidic technology, culture-expanded DPMSCs, isolated from the third molar teeth of adult donors, were fractionated into four subpopulations, each distinguished by distinct average cell diameters ranging from 14.3 to 20.5 μm. The medium-sized subpopulations (15.2 to 18.6 μm) demonstrated the highest colony-forming efficiency. In contrast, the large-sized subpopulation (18.6 to 20.5 μm) showed enhanced osteogenic and chondrogenic potencies in vitro, alongside superior abilities to suppress T cell proliferation and reverse macrophage M1 polarization in different co-culture settings. Furthermore, transcriptomic analysis revealed a progressively shifting gene expression profile with the change in DPMSC size. The large- and medium-sized subpopulation upregulates genes involved in immune responses, calcium signaling, and ECM-receptor interaction, while the small-sized subpopulation downregulates genes associated with immune response pathways, cell cycle, and growth factor activities. CONCLUSION: This study elucidates the morphological relevance of the functional heterogeneity of culture-expanded DPMSCs and proposes a viable strategy for the isolation of functional subpopulations of DPMSCs in clinical-scale manufacturing. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s13036-025-00524-w.