Purpose:
Transplantation of tissue engineered retinal pigment epithelial (RPE) cells has the prospect of becoming a widespread treatment option for blinding age-related macular degeneration. To achieve this goal, it is essential to establish a viable method for xenobiotic-free tissue banking of cultured RPE cells. In the present study, we measured the viability of human RPE cells after 7 days of xenobiotic-free storage at nine temperatures between 4° and 37°C
Methods:
Spontaneously immortalized ARPE-19 cells were seeded (5000 cells/cm2) on Nunclon Δ-surface multiwell plates and cultured in DMEM/F12 with 10% FBS until confluence. The cells were stored in HEPES-MEM at nine different temperatures (4°, 8°, 12°, 16°, 20°, 24°, 28°, 32°, and 37°C) for 7 days. For viability assessment, stored cells were incubated for one hour in PBS containing 1 µM calcein-acetoxymethyl ester (CAM) and 1 µM ethidium homodimer 1 (EH-1). CAM and EH-1 fluorescence was then measured by a microplate fluorometer. Mean fluorescence was calculated for all groups, and One-way ANOVA with Tukey’s post hoc comparisons were used for statistical analyses. Values presented are the mean of two separate experiments for all temperatures and three experiments for the three best temperatures, all performed in triplicates (n=6 and 12 cultures, respectively). P-values below 0.05 were considered significant.
Results:
Post-storage viability was dramatically reduced compared with unstored control cells. However, cells stored at 12°, 16° and 20°C differed markedly from the rest, with storage at 16°C (50 % survival) significantly enhancing viability compared with all other groups but 20°C (figure 1).
Conclusions:
The study demonstrates that storage temperature has a crucial impact on viability of RPE cells. We have identified a narrow temperature range (16°-20°C) that notably spurs cell survival. This study is the first step towards developing a suitable method for storage of RPE cells.
Keywords: retinal pigment epithelium • retinal culture • transplantation