APPLICATION NOTE: Using EVOM™ Auto ElectroZ to Monitor iPSC-RPE
Using Electrochemical Impedance Spectroscopy (EIS) to Monitor Confluency, Barrier Integrity, & Functional Polarity of Human iPSC-derived Retinal Pigment Epithelium (iPSC-RPE)
For decades, World Precision Instruments (WPI) EVOM™ technology has been trusted as the gold standard for transepithelial/transendothelial electrical resistance (TEER) measurements. EVOM™ instruments provide a reliable, non-invasive workflow for quantifying cell barrier integrity and permeability of cell cultures grown under physiological conditions, on semi-permeable inserts. The EVOM™ Auto ElectroZ builds on the established TEER workflow by adding Electrochemical Impedance Spectroscopy (EIS) capabilities, enabling researchers to gain deeper insights about their cells using the same, simple measurement workflow. EVOM™ Auto ElectroZ measures impedance across a broad frequency range from 2Hz to 100KHz, capturing additional information about cell growth, proliferation, morphology, differentiation, maturation, health, quality, and functional polarity. EIS measurements quantify TEER alongside additional parameters that provide deeper insights into cellular behavior. These include transepithelial/transendothelial capacitance (TEC), which provides information about cell confluency and morphology, and the membrane ratio (Tau ratio), which reflects electrical differences between the apical and basolateral cell membranes and serves as an indicator of functional cell polarity. By combining familiar TEER measurements with these additional EIS-derived parameters, the EVOM™ Auto ElectroZ enables researchers to develop a more comprehensive understanding of cell growth, structure, and function.
In this study, human induced pluripotent stem cell– derived retinal pigment epithelia (iPSC-RPE) were seeded at high and low densities on three commonly used, commercially available semi-permeable cell culture insert plate formats, including Corning 24-well HTS plates (Corning, Cat #3396), Corning 24-well inserts (Corning, Cat #3470) used with SUMILON companion plates (WPI, EVA-AC-03-03-12), and Corning 96-well HTS plates (Corning, Cat #3391). iPSC-RPE cells were seeded 45 days after differentiation and were allowed to grow and mature over the course of four weeks. iPSC-RPE cultures were maintained with regular media changes, and measurements were taken at three-day intervals with EVOM™ Auto ElectroZ, using the corresponding electrode array for each plate format (EVOM™ Auto ElectroZ Electrode Array C24, O24, and 96, respectively). Impedance measurements were taken at 66 frequencies from 2Hz to 50kHz and used to calculate TEER, TEC, and Tau ratio, which were monitored over the course of the experiment.
Across all three plate formats, TEER, TEC, and Tau ratio were measured with high repeatability, and values fell within the ranges reported by other comparable studies on biological cells grown in culture. TEC increased from approximately 1 μF/cm² on day 3 to 5 μF/cm² on day 15, before reaching a plateau. This trend is consistent with increasing cell coverage of the culture membrane as the cells approach confluence. As expected, TEER values remained relatively stable before Day 10, increasing slowly until day 15, and then continued to rise after day 15, reaching over 800 Ω-cm2 on day 25 across all three plates (Fig. 1 A-C).
TEER and TEC Measurements from Cell Seeding to Barrier Maturation. (A-C) Time course of TEERcellular (top) and TEC (bottom) during growth and maturation of iPSC-RPE cell cultures measured with EVOM™ Auto ElectroZ Electrode Arrays O24(A), C24(B), and 96(C) over day 2-26 after seeding on semi-permeable membranes. Dark blue and red indicate the higher seeding density, while light blue and orange indicate lower seeding density. Data are shown as mean ± standard deviation for biological replicates. (D): Rendering of the three electrode array types and their compatible cell culture plates (Corning 24-well HTS plates, Corning 24-well inserts, and Corning 96-well HTS plates).
TEER measurements increase as the barrier forms and matures over the course of the experiment, which is expected as iPSC-RPE establishes a confluent monolayer and tight junctions develop between the epithelial cells, forming tight barriers. Together, these trends indicate that barrier tightening initiates as the monolayer becomes confluent and morphologically stable, and continues to tighten after confluency is reached, a distinction that TEER and TEC together can reveal, but a single TEER reading cannot. Integrating EIS measurements throughout this workflow provides researchers with valuable insights into cell behavior and growth before barrier formation, followed by quantitative assessment of barrier integrity as the barrier develops and matures.
The Tau ratio (τ ratio, membrane ratio) is defined as the ratio of the fitted electrical time constants (Tau = R * C) associated with the apical and basolateral surfaces of an epithelial monolayer. The Tau ratio is a unitless value that reflects the membrane asymmetry and functional polarization of the cells. In previous studies, we have shown that the Tau ratio increases as epithelial cells differentiate and mature. Here, we monitored the Tau ratio of iPSC-RPE cells cultured in the Corning 96-well HTS plate format from cell seeding through barrier maturation over four weeks (Fig. 2). The Tau ratio of the high-density group increased initially, then plateaued around day 14 post-seeding. The low-density group followed the same trend for the Tau ratio, however, they were delayed by approximately five days, peaking around day 19 post-seeding. Furthermore, the magnitude of the Tau ratio in the high-density group stabilized around 5, almost half of that of the low seeding density group (~10). These results suggest that seeding density influences the kinetics of iPSC-RPE cells development and functional polarization.
Time course of Tau ratio during growth and maturation of iPSC-RPE from day 2-26 measured with EVOM™ Auto ElectroZ 96 Electrode Arrays. Dark green indicates the higher seeding density, while light green indicates the lower seeding density. Data are shown as mean ± standard deviation for biological replicates.
In summary, we demonstrate that EVOM™ Auto ElectroZ utilizes EIS technology to quantitatively measure TEER, TEC, and the Tau ratio, providing complementary, non-redundant biomarkers of cell growth, differentiation, maturation, and barrier function. While traditional TEER measurements can quickly assess barrier integrity following barrier formation, they may provide limited insight into cellular behavior during earlier stages of culture. By integrating TEER, TEC, and the Tau ratio, EVOM™ Auto ElectroZ enables researchers to monitor cellular development from initial growth and proliferation through differentiation, barrier formation, and functional maturation, providing a more comprehensive understanding of cell behavior throughout the culture workflow.
Acknowledgments
Data provided by Dr. Kapil Bharti’s laboratory at the National Eye Institute (NEI) under a Collaborative Research and Development Agreement (CRADA) with WPI.
References
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