Z6 - Label-Free Enrichment of Adipogenic hMSC Subpopulations Using Insulating Dielectrophoresis

Introduction: : Human mesenchymal stem cells (hMSCs), while useful in many therapeutic treatments, often struggle with producing consistent results in clinical trials. This is due to the inherent heterogeneity of hMSCs. To address this, many laboratories have used existing tools to sort hMSCs based on surface marker expression. Many of these techniques rely on fluorescent staining of biological markers, underscoring the need for label-free approaches that preserve the cells’ native state by leveraging intrinsic properties for sorting. Dielectrophoresis (DEP) is a label-free technique that sorts cells based on their inherent electrical properties when exposed to non-uniform electric fields, with cell behavior modulated by voltage and frequency. Using DEP, hMSCs can be separated based on their electrical properties to form subpopulations with enhanced differentiation potential for a given cell lineage.

Materials and

Methods: : In this study, we used an insulating DEP microfluidic device containing a large region of insulating posts to generate spatially non-uniform electric fields. The experiments were conducted on adipose tissue derived hMSCs that were grown to 80% confluency and utilized at passages 5 and 6. Prior to cell sorting, the passaged hMSCs were suspended in a DEP buffer with a conductivity of 100 µS/cm, and a concentration of 1 x 106 cells/mL. Under specific voltage-frequency combinations, a subset of cells became trapped at the insulating posts while others continue to flow through the device. By tuning these parameters, we enriched subpopulations of adipose tissue derived hMSCs that exhibited distinct DEP responses. Following sorting, the enriched cell populations underwent a 14-day adipogenic differentiation protocol.

Results, Conclusions, and Discussions:: The insulating DEP device uses a trap-and-release method for cell sorting. When DEP forces are present, untrapped cells are collected from the device, then the electrodes are switched off to collect trapped cells. Due to the impact of these DEP forces, the trapped cells have a slower velocity than the untrapped cells within the microfluidic device. Cells that flowed through the device (remained untrapped) under low-voltage, low-frequency conditions demonstrated enhanced adipogenic potential compared to a heterogeneous control population of hMSCs. Our work differs from previous cell sorting studies because we enrich hMSC subpopulations prior to differentiation, rather than sorting differentiated cells from a mixed population of various cell types. These findings demonstrate that DEP can be used to isolate functionally distinct hMSC subpopulations, offering a promising tool to address inherent cellular heterogeneity and enable targeted differentiation for regenerative therapies.

Acknowledgements and/or References (Optional):: I would like to thank UCOP for granting me the UC-HBCU Fellowship for my PhD studies, and NSF CAREER Award under grant 2048221 for funding this work.