Abstract
Purpose :
Retinal pigment epithelial (RPE) cells sustain photoreceptor integrity, and when this function is disrupted, retinal degenerations ensue. The purpose of this work is to produce and characterize cultures of human RPE cells that maintain desirable features to use them as tool for the study of the Retinal pigmen epithelium in vitro.
Methods :
We use healthy human 19-year-old donor eye cup from the human eye bank NDRI, complying with the ethical regulations for human donor subjects. The eye cups were dissected and explants were cultured in petri dishes, and the cells attached were then trypsinized and expanded in the several passages used for this work. ABC cells were characterized using immunocytochemistry for b-catenin, MITF and peropsin. Cells were cultured in trans-well system and determined the passive diffusion of trypan blue after reaching confluency, and markers of RPE were determined by qPCR and western blot assay. The phagocytosis was determined using photoreceptor outer segments (POS) or polystyrene microspheres (beads) to compare ABC cells to ARPE-19. RNAseq was performed to determine ABC uniqueness by comparing its transcriptome to ARPE-19 and a primary culture of a healthy 56-year-old male donor.
Results :
The newly characterized cell line from human RPE that we termed ABC remarkably recapitulates human eye native cells. They formed microvilli, tight junctions, and honeycomb packing and expressed distinctive markers. Outer segment phagocytosis, phagolysosome fate, phospholipid metabolism, and lipid mediator release were tested and showed resemblance with the human RPE can be studied. ABC cells displayed higher resistance to oxidative stress and are protected from senescence through mTOR inhibition, making them more stable in culture.
Conclusions :
ABC gene expression profile and phenotype displayed close proximity to native RPE lineage, making them a reliable cell system to unravel signaling in uncompensated oxidative stress and retinal degenerative disease to define neuroprotection sites.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.