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Robert F Mullins, S Scott Whitmore, Kathleen R Chirco, Grefachew Workalemahu, Shemin Zeng, Adam P DeLuca, XiuYing Liu, Jessica A Penticoff, Luke A Wiley, Edwin M Stone, Budd Tucker; The human RPE-choroid in short term organ culture: evaluation of gene expression changes. Invest. Ophthalmol. Vis. Sci. 2017;58(8):619. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
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.
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.
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.
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.
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