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Nathan Hotaling, Mylene Simon, Mohamed Ouladi, Nicholas Schaub, Tarreq Uddin, Praveen Manickam, Davide Ortolan, Helen Zhao, Joe Chalfoun, Peter Bajcsy, Kapil Bharti; REShAPE – A Cloud-Computing Based Cell Morphometry Analyzer for Development of a Release Criteria of Clinical Grade Retinal Pigment Epithelium. Invest. Ophthalmol. Vis. Sci. 2017;58(8):396.
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© ARVO (1962-2015); The Authors (2016-present)
One major challenge in developing a cell therapy product is development of release criteria that can define batch-to-batch variability, is consistent with product potency, and is easy to perform in a clinical setting. Here we develop an open access, cloud computing based, automated image analysis platform, REShAPE, that can perform morphometric analysis of retinal pigment epithelial (RPE) cells. We tested the ability of this platform to analyze good-laboratory-practice-grade (GLP-grade) induced pluripotent stem cell derived RPE cells from patients who had age-related macular degeneration (AMD iPSC-RPE). Furthermore, we compare the performance of REShAPE in analyzing millions of cells in images to that of desktop software.
Human iPSC derived RPE were cultured on nanofiber scaffolds or porous membranes. Cells were stained with anti-ZO-1-FITC to identify cell borders. Cell phenotype was assessed using transepithelial electrical resistance (TEER), gene expression, and ELISA based VEGF secretion in the supernatant. Cell morphometric quantification on over 100,000 cells/replicate x 2-5 replicates/RPE line x 8 RPE lines were analyzed. More than 30 shape metrics were used to describe the cells. The total time to analyze all images was compared between REShAPE and an ImageJ plugin installed on a standard desktop.
RPE differentiated from eight iPSC clones derived from three AMD patients had TEER values of more than 500 ohms/cm2 and demonstrated polarized VEGF secretion with higher amounts on the basal versus apical side. All GLP-grade AMD iPSC-RPE cells had a median of 6 neighbors and a hexagonality score between 7.5 and 8.5 out of 10 with non-significant variability between different batches of cells. The time to analyze images varied based on number of computing cores in the server but could be scaled to reach >100x faster than a standard desktop (>6 hours/image vs ~3 min./image). The software was also able to analyze subcellular features and could be run from multiple locations/computers simultaneously via a simple web browser.
The REShAPE software is an open access tool that provides ease of use and powerful data analytics. This tool helped define morphological features of GLP-grade patient RPE cells and provides potential release criteria to define identity, potency, and batch-to-batch variability of clinical-grade iPSC-RPE.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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