Technical Reference #1634

1634.

Effects of 400 nm; 420 nm; and 435.8 nm Radiations on Cultured Human Retinal Pigment Epithelial Cells Hyun-Yi Youn; Ralph Chou; Anthony Cullen; Jacob Sivak, University of Waterloo, Journal of Photochemistry and Photobiology B: Biology, 95(1634), (2009)

Abstract:
The present study demonstrates narrowband short-wavelengths radiation- (400 420 and 435.8 nm)induced cellular damage of cultured human retinal pigment epithelial cells using in vitro biological assaysto determine wavelengths that are responsible for

Keywords:
short-wavelength light radiation; RPE cell culture; cellular viability; mitochondrial damage; DNA damage; In vitro bioassays

Materials & Methods:
2.1. Human retinal pigment epithelial cell culture An immortalized human RPE cell line ARPE-19 was obtained from the American Type Culture Collection (Manassas VA USA) and cultured in DMEM/Ham’s F12 with L-glutamine and 15 mM Hepes (Media Tech VA USA) supplemented with 10% fetal bovine serum (Hycolone UT USA) and insulin–transferrin–sodium selenite (ITS) (Sigma MO USA). The cells were incubated in a humidified atmosphere of 5% CO2 and 95% air at 37 C and the culture medium was changed every 24 h. Cells were plated in T75 or T150 flasks (Falcon NJ USA) and allowed to reach confluence (typically 2–3 days). Glass bottom Petri culture dishes (MatTek MA USA) as well as 48-well plates (Falcon NJ USA) were used. Subculturing was carried out using a Trypsin/EDTA solution (Cascade Biologics OR USA) after a confluent monolayer appeared.2.2. Narrowband light exposure of cultured RPE cells The treatments tested were the visible light of wavelengths at 400 nm 420 nm and 435.8 nm. The wavelengths tested were chosen on the basis of commercially available interference filters. Irradiation sources were a Photochemical Research Associates (PRA) integrated arc lamp system using water cooled 1000W high pressure xenon arc in reflector fixtures (PRA Inc ON Canada). Fig. 1 shows a schematic diagram of the light irradiation apparatus. A front surface mirror was used to deflect the beam by 90 to impinge on the cells. A quartz-condensing lens (Edmund Scientific Co. NJ USA) was placed at the entrance and another quartz-condensing lens was placed at the exit aperture of the mirror housing respectively. Specific narrow wavebands (400 420 and 435.8 nm) of light were obtained using interference filters (Melles Griot NY USA). Table 1 shows the general characteristics of each filter. The arc lamp and optical system were enclosed and purged with nitrogen gas to prevent ozone formation before each use. Before each irradiation of cells dosimetry was performed using an 88XL Photodyne radiometer together with a photometric sensor head (Optikon Corp. ON Canada). To convert the radiometer measurement (lW) to irradiance (W/cm2) the measured value was divided by the photometric sensor head area (cm2) multiplied by the area irradiated on the cells (cm2) and multiplied by the linear multiplication factor (unit less) from the calibration curve. The sensor head area was 0.42 cm2 and the irradiated area was 5.30 cm2. The linear multiplication factors were 2.67 for 400 nm 2.42 for 420 nm and 2.23 for 435.8 nm respectively. For example a radiometer reading of 46 lW for 400 nm would be 0.00155 W/cm2 as irradiance (e.g. 46 lW/0.42 cm2 2.67 5.3 cm2 = 0.00155 W/ cm2). Radiant exposure time (s) was determined using the following radiometric equation: t = Hk/Ek where t is the exposure duration (s) Hk is the radiant energy level (J/cm2) and Ek is the measured irradiance (W/cm2). Table 2 shows the calculated energy levels and exposure durations for this experiment. Radiant exposure time was controlled with a preset electronic counter which automatically closed the shutter after each predetermined exposure. After 2–3 days of pre-incubation to form a confluent cell monolayer the cells were exposed to predetermined energy levels of light (see Table 2). The light source was positioned directly above the cells. In order to minimize the absorption of the radiation by the phenol red in the medium a thin layer of medium (about 1.0 mm) was left for only exposed cell groups and thus a minor phenol red effect may have influenced the results. Immediately after exposure cells were further incubated for 3–48 h under normal conditions (culture medium 37 C 5% CO2 95% air) before analysis. 2.3. Alamar blue assay After light exposure the fluorescent indicator dye Alamar blue (Medicorp PQ Canada) was used to evaluate cell viability. Alamar blue was diluted into the culture medium to 8% (v/v) and the solution was prepared immediately before each use to avoid possible precipitation. For experimental use cells were seeded into sterile flat-bottomed 48-well plates and cell density was adjusted to 1 105 cells/ml. Cells were allowed to settle and form a confluent monolayer for 2–3 days in their normal growing condition (culture medium 37 C 5% CO2 95% air) before being exposed to the light. Twenty four hours after irradiation cells were rinsed once with culture medium and 100–150 ll of 8% Alamar blue working solution was added to each well. Cells were further incubated for 1 h to allow the dye to be taken up by the cells. Fluorescent measurements were performed with the CytoFluorTM II fluorescence multiwell plate reader (PerSeptive Biosystems MA USA). The excitation/emission wavelengths settings were adjusted to 530/ 590 nm with the sensitivity gain set at 50 and temperature at 37 C.

Microscopic Technique
Confocal Microscopy, Laser Scanning

Cell Type(s)
ARPE-19