Introduction: : Cellular heterogeneity, driven by genetic, epigenetic, and environmental factors, plays a crucial role in processes like development, immune response, and disease progression, yet remains challenging to quantify due to the lack of standardized markers. Human mesenchymal stem cells (hMSCs), widely used in regenerative medicine for their multipotency and immunomodulatory properties, exemplify this complexity due to variations in morphology, size, and differentiation potential. Dielectrophoresis (DEP), a label-free technique that manipulates cells based on their dielectric properties, has emerged as a promising tool to characterize cell subpopulations, enhancing our understanding of heterogeneity. DEP occurs when a particle is introduced to a non-uniform electric field. By analyzing the DEP spectrum—specifically the transient slope at low frequencies—researchers can quantify cellular heterogeneity, with lower slopes and higher standard deviation indicating more heterogeneous populations. In this study, transient slope was analyzed across various cell types, including homogeneous HEK-293 cells and heterogeneous hMSCs from multiple tissue sources as well as cancer cells.
Materials and
Methods: : Various cell lines including HEK-293, hMSCs from adipose tissue (AT), bone marrow (BM), and umbilical cord (UC), as well as PC3 and DU145 cancer cells, were cultured under specific conditions and analyzed to assess cellular heterogeneity. hMSCs underwent osteogenic and adipogenic differentiation, and transient slope characterization was performed using a 3DEP analyzer to evaluate the DEP responses over a range of frequencies. Cells were prepared in a sucrose-glucose DEP buffer, and only high-quality DEP spectra (R² ≥ 0.9) were included in the analysis, with transient slope determined by fitting a linear trendline to the rise portion of the DEP spectrum. Additional assessments included quantifying cell morphology and size from microscopy images, and quantifying stemness with immunostaining for SOX2 and NANOG. Statistical comparisons were conducted using one-way ANOVA with Tukey’s post hoc test.
Results, Conclusions, and Discussions:: This study demonstrates the effectiveness of transient slope, derived from the DEP spectrum, as a label-free metric for assessing cell population heterogeneity. By comparing heterogeneous hMSCs to more homogeneous populations like HEK-293 and cancer cells (PC3 and DU145), we established that transient slope correlate with cellular diversity—lower slopes indicating greater heterogeneity. Two estimation methods were validated using various buffer conditions, yielding consistent results. The methods involved fitting a linear trendline to the low frequency region of the DEP spectrum: one using data from 10kHz to 20 MHz and the other from 2 kHz to 250 kHz. The smaller frequency range allowed fitting to more data points. Differentiation of hMSCs into osteoblasts and adipocytes resulted in higher transient slopes, reflecting lower heterogeneity. Compared to conventional techniques such as flow cytometry and RNA sequencing, transient slope offers a rapid, cost-effective, and marker-free alternative. While limitations exist—such as the need for single-cell validation and broader cell type inclusion, the findings support transient slope as a promising metric for assessing cell population heterogeneity. Transient slope derived from the DEP spectrum offers a promising, label-free approach for assessing cell population heterogeneity. This study presents key advancements, including two methods for implementing transient slope, its successful application in characterizing heterogeneity in both stem and cancer cells, and its demonstrated sensitivity.
