Flow-based microfluidic-chip filtration for efficient sorting and evaluating differentiation potential of human mesenchymal stem cell subpopulations

• Model framework of hydrodynamic filtration for trimodal separation of hMSCs. • Well-designed microfluidic-chip filtration isolates high-potency hMSC subpopulation. • Small RS cells exhibit the highest multilineage potential and self-renewal capacity. • Label-free and continuous sorting of hMSC enhances therapeutic efficacy. Human bone marrow-derived mesenchymal stem cells (hMSCs) comprise diverse subpopulations with different levels of multipotency, which refers to the capability of stem cells to differentiate into a limited range of cell types. Notably, smaller cells typically exhibit greater multipotency, whereas larger cells show reduced differentiation potential. Identifying and isolating the highly multipotent small subpopulation from this heterogeneous mixture is crucial for enhancing the efficacy of stem cell therapies. To achieve this purpose, we have successfully separated hMSCs into three subpopulations owing to differences in size by employing a properly designed microfluidic-chip based on the hydrodynamic filtration principle. This microfluidic-chip, designed with the parameters accurately determined by the full analysis of laminar flow theorem in channel networks, enables the efficient and continuous sorting of small rapidly self-renewing (RS) cells (<25 µm), spindle-shaped (SS) cells (25–40 µm), and large flattened (FL) cells (>40 µm). Due to limited studies on these subpopulations, we evaluated their stemness properties and multipotency through adipogenic, osteogenic, and glial differentiation assays. RS cells showed the highest pluripotency marker expression, followed by SS, unsorted, and FL cells. Furthermore, analyses of differentiation efficiency and expression levels of differentiation marker genes demonstrated that both RS and SS cells exhibited significantly higher multilineage potential compared to FL cells, with RS cells showing the most pronounced differentiation capacity. These findings highlight the efficacy of our microfluidic-chip in precisely sorting target hMSC subpopulations with high multipotency and self-renewal capacity, supporting its potential to enhance stem cell-based therapeutic applications.