Abstract
Purpose: :
Culturing adult primary hRPE cells has proven challenging. These cells grow slowly, have a tendency to adopt a fusiform morphology, and detach in the presence of oxidative stress (OS). Thus in this study, we first established optimal culture conditions and then asked if docosahexaenoic acid (DHA) pretreatment induces protection against H2O2-mediated cell death, as seen in ARPE-19 cells.
Methods: :
Cells were cultured in Eagle Minimum Essential Medium 10% Fbs with FGF-β (10ng/mL) in the presence of Rho kinase inhibitor, retinoic acid, or Poly-L-Lysine coated plates. Once cuboidal morphology was attained, the cells were characterized with RPE cell markers ZO-1, RPE65, and Neu-N via Western blot and immunocytochemical analysis. All cellular culture conditions were tested using varying levels of OS. Using Poly-L-lysine coated plates, cells were pretreated with 600 nM DHA for 6h then placed in DHA free medium for 24h before challenge with H2O2+TNF-α. Apoptosis was assessed using Hoechst staining and confocal microscopy.
Results: :
hRPE cells cultured with Rho kinase inhibitor were most resilient to OS-induced apoptosis, yet they failed to achieve the cuboidal morphology characteristic of RPE cells. When Rho kinase inhibitor was combined with Poly-L-Lysine-coated plates, this effect was enhanced (J.Neuroscience.Res.,86,270-80,2008). Cells cultured on Poly-L-Lysine-coated plates without Rho kinase inhibitor achieved the epithelial-like morphology and were slightly more sensitive to OS than Rho kinase inhibitor alone, but this difference was not significant. In both the control and retinoic acid conditions, cells detached at 150µM OS. Western blot and immuncytochemical analysis of Poly-L-Lysine coated plates indicate that these cells express ZO-1, RPE65, and Neu-N. In addition, when challenged with 0, 150, 250, and 350 µM OS +TNF-α these cells displayed survival levels of 99.6±0.1% 95.5±1.5%, 69.6±12.5%, 59.2±8.8%, respectively, while DHA exhibited protection with survival levels of 97.6±0.9%, 96.3±0.9%, 95.4±3.4%, and 87.1±10.3% respectively.
Conclusions: :
Our results demonstrate that hRPE cells (expressing ZO-1, RPE65, and Neu-N), grown on Poly-L-Lysine-coated plates in the presence of FGF-β, result in high densities of cuboidal cells, which remain attached in the presence of OS, and provide better cell morphology than Poly-L-Lysine-coated plates with the Rho inhibitor. Also, when pretreated with DHA, these cells are protected from H2O2-induced cell death, implicating the importance of DHA levels in hRPE cell survival.
Keywords: lipids • oxidation/oxidative or free radical damage • retinal culture