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Colby Foster Lewallen, Qin Wan, Arvydas Maminishkis, William Stoy, Ilya Kolb, Nathan Hotaling, Kapil Bharti, Craig R. Forest; Towards automated intracellular electrophysiology of retinal pigment epithelium. Invest. Ophthalmol. Vis. Sci. 2019;60(9):1938.
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© ARVO (1962-2015); The Authors (2016-present)
An induced pluripotent stem cell-derived retinal pigment epithelium (iPSC-RPE) patch, transplanted in the subretinal space, has recently demonstrated functional recovery of damaged RPE in a pig model. A protocol for quantifying iPSC-RPE-patch health and function prior to transplantation is a critical need before this therapy reaches the clinic. Intracellular electrophysiology is such an assay, but it currently requires extensive training and skills that make it impractical for use in the clinic. Therefore, in this study, we aimed to automate RPE intracellular electrophysiological measurements.
iPSC-RPE were seeded on 12-well, semi-permeable Transwell inserts and cultured for 4 to 6 weeks prior to analysis. RPE were then placed into an Üssing chamber that was modified to allow separate apical and basal membrane perfusion and intracellular access with a sharp microelectrode. Transepithelial potential was measured with agar bridges in series with calomel electrodes. Basolateral membrane potential was measured with the microelectrode. Microelectrode position was controlled by a combination of 3-axis micromanipulator and linear stage. To study cell physiology in response to physiological stimuli, the normal Ringer solution was changed to Ringer with either 1 mM reduced-potassium, or 100 µM ATP.
We developed an algorithm that automatically, and without human intervention, inserts a sharp microelectrode into iPSC-RPE with a 3x higher efficiency than gold standard, human operators (28% vs 10% success). Indention depth (60% of RPE thickness), electroporation duration (100 µs), electroporation depth (83% of indention depth), and cell mounting surface thickness (245 µm) were optimized to maximize recording yield. The algorithm enables stable and rapid microelectrode placement into the cytoplasm of RPE for standardized intracellular electrophysiological measurements.
An optimized algorithm for inserting a sharp microelectrode during intracellular electrophysiology of RPE is presented. This algorithm simultaneously increases successful recording yield and reduces required experimenter effort and skill. This algorithm can be utilized to study membrane properties of RPE, ion channels, and various intracellular signal pathways; essential for evaluating iPSC-RPE-patch potency.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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