Purchase this article with an account.
M Natalia Vergara, MIGUEL FLORES-BELLVER, Silvia Aparicio Domingo, Minda M. McNally, Karl J Wahlin, Meera Saxena, Jeff S Mumm, Valeria Canto Soler; Development of an automated platform for large-scale fluorescence screening of 3-D retinal organoids. Invest. Ophthalmol. Vis. Sci. 2017;58(8):4556.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
The development of stem cell-derived retinal organoid systems has opened previously unimaginable possibilities for the study and potential treatment of retinal degenerative diseases. However their variability and the lack of high throughput platforms for their analysis pose severe limitations for their use in drug development applications. Hence, the purpose of this work was to develop a platform (3D- ARQ) that enables fluorescence quantification-based large-scale drug screening in complex human iPSC-derived retinal organoids, while providing the speed, sensitivity and reproducibility necessary for this kind of assays.
Transgenic hiPSC lines harboring GFP or YFP fluorescent reporters were generated and induced to differentiate into retinal organoids. Wild type retinal organoids were stained with various fluorescent dyes and used for parameter optimization purposes. Non-stained organoids were used as controls in all experiments. Organoids were seeded into v-bottom 96 well plates and scanned using a fluorescence microplate reader that allows XYZ-dimensional detection and fine-tuned wavelength selection. Results were confirmed byconfocal microscopy.
Using this approach we: i) optimized the parameters for detecting fluorescent reporter signal in retinal organoids, ii) characterized autofluorescent background profiles, iii) determined the most favorable reporter emission ranges for 3D-ARQ-based quantification of retinal organoids, iv) established co-labeling methods to compensate for size variability between individual samples, and v) determined the quality of the assay for HTS applications by evaluating performance and sensitivity parameters. We validated the quantitative power of 3D-ARQ by measuring fluorescence intensity in global transgenic vs. chimeric YFP-expressing retinal organoids; demonstrated the applicability of this technology to track developmental progression by quantifying immunofluorescence staining for Otx2 at different weeks of differentiation; and exemplified the capacity of 3D-ARQ to track changes in physiological state by using the system to measure ROS accumulation following an oxidative insult.
We have developed and validated a technology that enables the rapid and robust quantification of fluorescence reporters in hiPSC-derived retinal organoids to a level that meets HTS requirements for drug discovery applications.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
This PDF is available to Subscribers Only