Downregulation of lysosomal trafficking in ARPE19 cells leads to decreased transfection efficiency at high passage

PURPOSEARPE19 cells are a commonly used cell culture model for the study of retinal pigment epithelial cell biology and pathologies. However, numerous studies have demonstrated that ARPE19 undergo morphologic, transcriptomic and genomic alterations over time and with increasing passage number. Herein, we explore the mechanisms underlying increased resistance to the delivery of exogenous genetic material via transfection in ARPE19 cells using mass spectrometry. METHODSARPE19 cells (N=5 wells/reagent) were seeded in 6-well plates at passages 24 through 30. At 70% confluency an mCherry reporter construct was delivered via transfection using Lipofectamine 3000, Lipofectamine LTX, Lipofectamine Stem, or PEI (polyethylenimine) reagents. After 72 hours, transfection efficiency was quantified by fluorescence microscopy and flow cytometry. Mass spectrometry and immunofluorescence of ARPE19 cells were performed at passages 24 and 30 to evaluate altered protein synthesis and localization between passage numbers. RESULTSARPE19 transfection showed a maximum transfection efficiency of 32.4% at P26 using Lipofectamine 3000 reagent. All lipofectamine based reagents demonstrated statistically significant decreases in transfection efficiency between passages 24 and 30. Mass spectrometry analysis revealed 18 differentially expressed proteins, including down-regulation of clathrin light chain B (CLTB) and legumain (LGMN) that was confirmed via immunofluorescence imaging, which indicated altered intracellular localization. CONCLUSIONSARPE19 cells demonstrate passage number dependent changes in lipofectamine-based transfection efficiency. Mass spectrometry and immunofluorescence indicates the observed decrease in transfection efficiency involves the dysregulation of endocytosis and intracellular endolysosomal trafficking at later passages. TRANSLATIONAL RELEVANCEThis study contributes to mounting evidence for changes in ARPE19 cell physiology with increasing passage number. This information is of value for the continued use of ARPE19 cells as a model system for RPE biology and the development of therapeutics.