Abstract
Purpose :
Age-related macular degeneration (AMD) is the leading cause of blindness in the elderly and is estimated to affect approximately 8.7% of all individuals between the ages of 45 and 85. In the early or 'dry' form of the disease, protein and lipid deposits, termed drusen, develop between the retinal pigment epithelium (RPE) and the selectively permeable barrier separating the RPE and retina from the choroidal circulation, called Bruch’s membrane, particularly within the macular region of the eye. Apart from aged non-human primates, drusen development is not easily replicated using animal models, however several studies have demonstrated that primary RPE cells, cultured in monolayers accumulate drusen-like deposits. Herein, we assess the accumulation of ‘drusen’ longitudinally in primary porcine RPE cultures and characterize the protein and lipid content of individual drusen to establish their similarity to drusen recovered from human donor eyes with diagnosed AMD.
Methods :
Fresh (< 6 hours post mortem) porcine eyes were obtained from a local abattoir and disinfected through immersion in PVP prep solution. On ice, the cornea, lens, vitreous body and retina were removed mechanically before the primary RPE was isolated via enzymatic dissociation using serial incubations in 0.25% trypsin in L-15 media at 37°C. Cells were incubated in 1% DNase I for 2 minutes, purified via centrifugation on a 40% Percoll cushion with 0.01 M Na2PO4 and 0.15 M NaCl (pH 7.4), and plated directly on polyethylene terephthalate (PET) transwell membranes. RPE cultures were maintained for up to 26 weeks without passaging. Transwell inserts were fixed in 4% PFA and cryosectioned or flat mounted. Drusen components, size, and quantity were analyzed via immunofluorescence microscopy.
Results :
Freshly isolated RPE take approximately 5 – 6 days to fully anchor to the membrane, after which cells no longer slough off with feeding. Throughout the 26 week aging period, approximately 60% of the cultures show no signs of infection. As young as 22 weeks post isolation, primary RPE cultures developed deposits with drusen-like characteristics such as lipid accumulation indicated by Nile Red and positive immunofluorescent staining for known drusen components such as ApoE, vitronectin, and TIMP3.
Conclusions :
Through this study, we further verify the efficacy and reproducibility of primary RPE culture as a method of ex vivo modeling of drusen-formation.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.