Abstract
Purpose :
Conventional methods for studying the membrane electrophysiology of RPE cells, such as channels, transport mechanisms, or morphology are complex and invasive. The purpose of this study was to develop and validate a non-invasive method using electrochemical impedance spectroscopy (EIS) that enables the collection of unique, microelectrode-like data.
Methods :
A mathematical model with a penalty function was developed. The penalty function relies on a user-defined ratio of transcellular to paracellular resistance (junction resistance ratio, JRR). We tested our model using JRRs between 0.1 and 10 on RPE EIS measurements between 1 Hz and 10 kHz in an Ussing chamber with continuous perfusion. Validation of our method was performed by using an invasive microelectrode method that measures the distinct resistance and capacitance values of each transport barrier. The response of RPE to the apical application of 100 µM adenosine triphosphate (ATP) was simultaneously recorded using our new method and the previously validated, microelectrode method.
Results :
The JRR for our RPE cells, based on the intracellular electrode data, was 5 (SD 1, n=7). During ATP stimulation, the intracellular electrode method shows a concomitant increase in apical resistance and decrease in basolateral resistance in our RPE. Our EIS-based method reveals similar TER and directional changes in each membrane response during apical ATP application. Variation of JRRs between 1 and 10 show similar responses, demonstrating robustness to JRR inaccuracy.
Conclusions :
This method offers a higher-content, longitudinal, and non-invasive assessment of RPE physiology. Our JRR method can replace the need for the invasive microelectrode method. Our JRR method yields an excellent approximation of membrane-specific changes. This method will significantly enhance drug screening and RPE-cell QC, especially in studies targeting membrane-specific responses of epithelial cells, not just RPE.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.