Introduction: : Understanding heterogeneity within patient derived stem cell populations can help lead to more effective and accurate modeling for a variety of diseases in vitro, as it can influence cell behavior leading to inconsistent results and complications. Therefore, this presentation focuses on exploring heterogeneity within stem cell populations using electrokinetic approaches. Specifically, dielectrophoresis (DEP) is explored as a label-free method of manipulating, characterizing and separating cell populations through the application of a non-uniform alternating current (AC) electric field.
Materials and
Methods: : Dielectrophoretic manipulation occurs when applying a voltage at a set frequency, cells will either move towards the electric field gradient or be repelled from the electric field gradient, referenced as positive DEP and negative DEP, respectively. Two main modes of DEP used in this work are traditional/electrode-based DEP and insulating-based DEP (iDEP). Traditional DEP uses metal electrodes and are used in my work to characterize the cells. Specifically, the 3DEP analyzer is used, which consists of twenty microwells each at their own frequency, which can be used to model the behavior of cells at different frequencies. The dielectric properties of the cells can be extracted (e.g. cell membrane capacitance, conductivity and permittivity) correlating to unique properties of the cell. IDEP employs an array of posts from insulating material (e.g. glass, plastic, or polydimethylsiloxane), to generate a non-uniform electric field and is used for cell separation. A continuous flow trap and release method is used for separation, where cells experiencing positive DEP are trapped along the columns, while cells experiencing negative DEP continue to flow, and both populations are collected for characterization. Parameters such as voltage, frequency and electrode configuration can be used to tune trapping, and generate strategies for optimal cell separation, for downstream cell characterization analysis. Combination of these electrokinetic techniques allow for both quantification of their dielectric properties and enrichment of desired cell populations, to better improve the application of patient specific modeling in research.
Results, Conclusions, and Discussions:: The two populations of stem cell derived sources explored in my work are adipose tissue (AT) derived human mesenchymal stem cells (hMSCs) and human induced pluripotent stem cell (hiPSC) derived cardiomyocytes (CMs). For hMSCs, the varied differentiation potential within a single source lends to their inherent heterogeneity and creates challenges for applying hMSCs for therapeutics. With the iDEP device, the two populations (trapped and untrapped), can be generated based on the electric field conditions. After a 14-day adipocyte differentiation, histological staining of the untrapped populations show variation with the separated populations can vary efficiencies for adipocyte cell generation. Human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) are explored in this work due to the challenge of overcoming their immaturity and impurity post-differentiation, which can significantly affect the accuracy of cellular function and behavior. The application of traditional DEP is useful for gaining information regarding the electrical properties of hiPSC-CM populations and comparing it to more adult-like CM and undifferentiated hiPSC models, for a measure of the extent of maturation. IDEP is being utilized to generate enriched CM populations to show the capability of DEP-based platforms as well as compare the performance of homogeneous and heterogeneous population in response to light-based photostimulation and its influence on maturation. Current results for the electrical properties have shown similarities between differentiated and undifferentiated hiPSC populations and significant differences between adult rat CMs and hiPSC-CMs, which can be indicative of the immaturity and heterogeneity that exists within hiPSC-CM populations. Overall, my work examines how DEP can be applied as a technique to (i) investigate the heterogeneity within stem cell populations through quantification of cellular dielectric properties and (ii) generate enriched populations of target cell types. In the future, I envision that these platforms will pave the way for label-free and real-time technologies that can allow for patient specific and effective development of in vitro models for clinical applications.
