The human RPE-choroid in short term organ culture: evaluation of gene expression changes

Abstract

Purpose : Robust in vitro systems, employed to study the effects of disease-associated environmental insults on cellular physiology, offer excellent opportunities to understand the pathology of diseases such as age-related macular degeneration. Previous studies by us and others have shown ultrastructural preservation of RPE-choroid organ cultures after several days. In the current study we compared gene expression in freshly isolated and cultured tissues and cells.

Methods : Human donor eyes were obtained and utilized for: (a) RPE isolation, followed by either flash freezing (n=3 samples, donor 1) or 7 days incubation on tissue culture plastic (n=3 samples, donor 1); or (b) RPE-choroid collection, followed by flash freezing (n=3 samples, donor 2) or 7 day organ culture (n=3 samples, donor 2). Total RNA was then isolated and transcript levels were determined using RNA-Seq (75 nucleotide paired-end reads of poly-A selected RNA). Sequencing reads were mapped to the human genome build hg19 using Tophat2 and quantified using Rsubread. Differential expression analysis was performed using edgeR.

Results : RPE cells in organ cultures showed more similarity to the RPE in vivo than did RPE cells that were plated on tissue culture plastic. When expression of the RPE-specific genes RPE65, BEST1, and RLBP1 was evaluated, levels in isolated RPE cells on tissue culture plastic were reduced to 2%, 3% and <1% of uncultured cell levels, whereas in organ cultures these levels were 65%, 83% and 25% of flash frozen samples, respectively. Cultured RPE-choroid also continued to express vascular endothelial cell-specific genes VWF, CDH5, CD34, and ICAM2 after 7 days of culture at similar levels to fresh tissue.

Conclusions : Human RPE-choroid organ cultures have relatively high fidelity of gene expression compared to primary cultures and retain viable RPE and endothelial cells at least up to 7 days in culture.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.