Use of ARPE-19 cell line as an in-vitro model for age related macular degeneration

Abstract

 

Purpose

 

Age-related Macular Degeneration (AMD) is the most common cause of blindness in the elderly in the western world. One of the earliest signs of AMD is the formation of drusen- extracellular deposits beneath the Retinal Pigmented Epithelium (RPE) cells which are the prime target of the disease. The sequence of biochemical, cellular and molecular events leading to the development of dry form AMD is still poorly understood mainly because of the lack of appropriate models. This study describes the production of Sub-RPE deposits in cultured ARPE-19 cell line.

 

Methods

 

Early passage (P 7-12) Human adult retinal pigment epithelial cells (ARPE-19) were seeded onto laminin-coated porous supports. Cells were cultured in 10% DMEM/Ham’s F-12 medium then switched to serum free medium. After 3 months in culture, cells were exposed to complement-competent human sera at 10% (vol/vol) in serum-free DMEM/Ham’s F-12 medium for 24h then fixed in 4% PFA. Immunofluorescence (IF) analysis was used to determine the immunoreactivity of several drusen components in the sub-RPE deposits. For western blot (WB) analysis, cells were seeded in 6 well plates. Cells were cultured in DMEM/Ham’s F12 as above for 48h then serum starved and cultured for 3 months before analysis.

 

Results

 

IF analysis showed signs of aged RPE morphology in serum free cultures compared to 10% FCS. Serum free cultures grown on porous supports had larger nuclei that stained negative for the cellular proliferation nuclear protein KI-67. There was also a significant increase in lipid accumulation when cells were stained with Nile red (P = 0.0013). Serum free cultures also showed the presence of small spherical deposits that accumulated within the porous support material. WB and IF analysis for markers of complement activation and AMD pathology showed differences between serum starved and serum fed cells.

 

Conclusions

 

These findings suggest the use of long term ARPE-19 cells cultured in serum-free medium as a promising model for drusen deposition. This would facilitate the analysis of molecular and cellular characteristics of AMD pathogenesis, augmenting the therapeutic strategies for dry AMD.

 

 

Sub-RPE deposits accumulation in three month old serum starved ARPE-19 cell culture. Immunofluorescence analysis showed the acummulation of sporadic deposits underneth the RPE monolayer that is immunoreactive for APOE antibody (green). (Scale bars, 25 µm in A and B; 10 µm in C).

 

Keywords: 412 age-related macular degeneration • 701 retinal pigment epithelium • 583 lipids