Abstract
Purpose: :
To evaluate the toxicity effects of the exposition of human retinal pigmented epithelium cultured cells (ARPE-19) to different solutions with varying degrees of osmolarity and pH.
Methods: :
ARPE-19 cultured cells were submitted to a 10 minutes exposition to balanced salt solution (BSS) manipulated solutions so as to obtain the following pHs: 3, 4, 5, 6, 7, 7.5, 8 and 9 (with osmolality range from 317 to 339 mOsm/kg). Moreover, ARPE-19 cultured cells were submitted to a 10 minutes exposition to glucose solutions at the following concentrations: 2.5%, 5%, 10%, 20%, 40% and 50% (pH adjusted to 7, resulting in the following osmolalities respectively: 142, 277, 573, 1156, 2410 and "more than 2500" mOsm/kg). Other experiment with the same methodology was performed with mannitol solutions at the following concentrations: 1%, 2.5%, 5%, 10% 15% and 20% (pH adjusted to 7, resulting in the following osmolalities respectively: 74, 150, 309, 464, 470 and 658 mOsm/kg). The toxicity was estimated through the MTT assay, a standardized colorimetric method used as a measure of viable (living) cells. Each solution was tested in at least three separate weils, and the arithmetic mean was considered for statistical analysis.
Results: :
All results obtained are described below as percentages of the MTT reading obtained with control BSS solution. Regarding pH: 3(40%), 4(50%), 5(84%), 6(100%), 7(100%), 8(71,5%), 9(38%). Regarding the glucose solution: 2.5%(82%), 5%(81%), 10%(70%), 20%(53%), 40%(30%) and 50%(28%)(SD±0.04). Regarding the mannitol solution: 1%(93%), 2.5%(96%), 5%(98%), 10%(100%), 15%(100%), 20%(100%) (SD±0.01).
Conclusions: :
Different dilution methods are applied to many dyes in vitreoretinal surgery. Much of the resultant toxicity may be related to the solution and not to the dye itself. Osmolarity and pH are important factors to be considered. Our study revealed that the safety window of pH in cultured RPE cells is between 5 and 7. Regarding osmolarity, glucose solution even at iso-osmolar ranges (280 and 320 mOsm/Kg) promoted some cell death. Regarding mannitol solutions there was no toxicity even in osmolality as high as 650 mOsm/Kg. Mannitol solution may be an alternative if hypertonic solution is needed in order to obtain dye decantation.