Human RPE ex vivo ‘Flatmount Technique’ for Comparative Morphometric and Tissue Culture Survival Analysis (Mouse) Using Alizarin Red Staining, Live/Dead Cell Analysis and Epifluorescent Microscopy

Abstract

Abstract: : Purpose: To measure the age influence on retinal pigment epithelium (RPE) morphology in human ‘flat–mounts’ and the ex vivo viability of mouse RPE ‘flat–mounts’ in tissue culture. Methods: We have previously described a RPE ‘flat–mount’ analysis technique in different species (ARVO abstract # 3678, 2004). Ten human eyes from different age groups (20 – 66 yrs) were stained with Alizarin Red S (1%) and analyzed by light microscopy. Retinal pigment epithelial cell density (RPECD) ± SD, coefficient of variability (CV) and hexagonality were determined after digital photography by using Konan KSS–300 analysis software. Measurements were also performed in corresponding corneal endothelial cells in humans (ECD). Live/dead cell counterstaining was used to determine the survival potential of ex vivo RPE ‘flatmounts’ from BalbC (n=4) and C57BL/6 mice (n=7) by fluorescent microscope from fresh specimens and from 5 day tissue cultured mouse RPE. Results: In a 20 year old human the foveal RPECD (mean±SD) was 6419±59 cells/mm², the central RPECD was 4090±86, and the peripheral RPECD was 2853±140, while ECD was 3703±99. In a 46 year old human the central RPECD was 3895±88, and the peripheral RPECD was 2552±172, while ECD was 2732±102. In a 66 year old human the central RPECD was 4839±98, the peripheral RPECD was 2370±171, while ECD was 2820±116. Human tissue analysis was dependent on a time limit of less than 20 hours after death. Highly pigmented tissue was not suitable for staining. Live/dead cell analysis of ex vivo mouse ‘flatmounts’ verified viable RPE cells up to five days in tissue culture. Conclusions: Micro dissected ‘flat–mounts’ of the RPE offer excellent possibilities to monitor for disease processes in many species including humans. The RPECD was highest in fovea, central density increased and peripheral density decreased with age. Ex vivo RPE ‘flatmounts’ in mice hold promise for possible future tissue culture and experiments over time.

Keywords: retinal pigment epithelium • retinal culture • cell adhesions/cell junctions