Abstract
Purpose: :
To evaluate the effects of MPA over two distinct human retinal cell lines in vitro: human retinal pigment epithelium (ARPE-19) and Human Muller Cells (type MIO M-1), and to determine a safety profile of MPA in vitro.
Methods: :
ARPE-19 cells (ATCC, Manassas, VA) and MIO M-1 cells (Dept. of Cell Biology of the University College, London, UK) were treated with 25, 50 and 100 µg/ml of Mycophenolate mofetil (Cellcept, Roche, Nutley, NJ) for 24 hours. The powder was mixed and diluted into culture media in order to achieve the desired concentration. Safety was evaluated by trypan blue dye-exclusion cell viability assay, JC-1 mitochondrial membrane potential (ΔΨm) assay, and caspase-3/7 apoptosis related assay.
Results: :
Both cell lines showed a significant reduction in cell viability at the highest dose after 24 hours (ARPE-19 cells: 69.75 ± 1.06%, 92.10 ± 1.84% and 91.90 ± 1.83% for 100, 50 or 25µg/ml, respectively versus 96.10 ± 0.42% for untreated controls; MIO M-1 cells: 74.80 ± 1.84%; 87.00 ± 2.26% and 89.02 ± 0.99% for 100, 50 or 25µg/ml, respectively and 90.35 ± 0.64% for untreated controls, p<0.001 for both cell lines). The caspase 3/7 apoptosis related assay showed a statistically higher activity for both cell lines at the 100µg/ml concentration (ARPE-19: 14248.38 ± 1833.37, 5418.44 ± 2583.94, and 3274.44 ± 1458.62 msi for 100, 50 and 25µg/ml respectively, versus 2705.03 ± 488.26 msi for untreated controls, p<0.001; MIOM-1 cells: 14405.03 ± 1339.51, 5649.29 ± 1269.98, and 4251.13 ± 3211.69 msi for 100, 50 and 25µg/ml respectively, versus 2593.33 ± 937.53 msi for untreated controls, p<0.05). The JC-1 mitochondrial membrane potential (ΔΨm) did not show statistical differences for both cell lines tested.
Conclusions: :
Doses of MPA up to 50µg/ml seem to be safe in vitro. This data must be validated by experimental models in vivo in order to confirm its safety for clinical use. Future experiments with autoimmune models should be performed as well to determine if local intraocular applications of MPA are effective in controlling inflammation.
Keywords: retinal culture • apoptosis/cell death • drug toxicity/drug effects