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Colby Foster Lewallen, Qin Wan, Arvydas Maminishkis, Kapil Bharti, Craig R. Forest; At-line functional characterization of iPSC-derived retinal pigment epithelium. Invest. Ophthalmol. Vis. Sci. 2020;61(7):4127.
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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 non-destructive, yet comprehensive, potency assay must be developed before this therapy reaches the clinic. Intracellular electrophysiology is the gold-standard method for evaluating RPE properties (e.g., cell polarity and membrane-specific resistances), but is destructive, costly, and difficult to perform. Therefore, in this study, we aim to leverage the simplicity of extracellular electrophysiology while overcoming its limitations by teasing apart RPE membrane-specific properties using electrochemical impedance spectroscopy (EIS).
iPSC-RPE are seeded on 12-well, semi-permeable Transwell inserts and cultured for 6-8 weeks prior to analysis. iPSC-RPE are placed into a commercially available device (EndOhm-12G) modified to perform galvanostatic EIS (MetrOhm). RPE capacitive responses to osmotic gradients are measured with EIS using hyperosmotic Ringer’s solutions created by the addition of mannitol. The relationship between intracellular, membrane-specific properties and a t-ratio (obtained with EIS) is measured by adding forskolin to the apical and basolateral baths.
We demonstrate that EIS has the unique ability to rapidly and non-destructively evaluate crucial properties of iPSC-RPE, at-line, as part of a comprehensive release assay for an upcoming clinical trial. Specifically, we quantify iPSC-RPE’s capacitive response to osmotic gradients and link intracellular electrophysiological data to a unique t-ratio parameter to provide novel data for comparison with healthy cell function prior to transplantation.
EIS can simultaneously add new quantitative metrics to a release assay and gain insights into cell properties previously only obtained utilizing destructive, intracellular techniques. EIS could become a platform for standardized assessment of potency for many other future stem cell therapies.
This is a 2020 ARVO Annual Meeting abstract.
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