RF/6A Chorioretinal Cells do not Display Key Endothelial Phenotypes

Purpose: The RF/6A chorioretinal cell line originally isolated from a fetal rhesus macaque in 1968 is widely used to model human retinal and choroidal endothelial cells in cell culture. We sought to determine the extent to which this line possesses endothelial cell-specific characteristics. Methods: RF/6A cells obtained from American Type Culture Collection were subjected to next-generation RNA sequencing. Transcriptomic-based cell type profiling was conducted using xCell. Validation of endothelial cell-specific marker expression was conducted by qRT-PCR and western blotting. Functional assays of acetylated low density lipoprotein uptake, TNF-?-induced adhesion molecule expression, shear stress alignment, and endothelial tube formation were assessed. Results: RF/6A transcriptomic profiles obtained de novo or from a publically available repository did not correspond to endothelial gene expression signatures. Expression of established endothelial markers (PECAM1, CDH5, KDR, VWF, ICAM2, ENG, or TEK) were very low or undetectable in RF/6A compared to primary human umbilical vein endothelial cells. Compared to primary endothelial cells, RF/6A were unable to uptake acetylated LDL, exhibit induction of E-selectin in response to TNF-? treatment, align in the direction of shear stress, or form regular capillary-like tubes in Matrigel. Conclusions: RF/6A do not exhibit key endothelial cell characteristics or behaviors. Therefore, caution should be employed in designing and interpreting studies using these cells as surrogates for choroidal or retinal endothelial cells. RF/6A cells obtained from American Type Culture Collection were subjected to next-generation RNA sequencing. Transcriptomic-based cell type profiling was conducted using xCell. Validation of endothelial cell-specific marker expression was conducted by qRT-PCR and western blotting. Functional assays of acetylated low density lipoprotein uptake, TNF-?-induced adhesion molecule expression, shear stress alignment, and endothelial tube formation were assessed.