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Shaun P Garland, Joshua Morgan, Christopher J Murphy, Paul Russell; Cell culture of trabecular meshwork cells under continuous oxidative stress by photocatalytic generation of H2O2. Invest. Ophthalmol. Vis. Sci. 2014;55(13):2905.
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© ARVO (1962-2015); The Authors (2016-present)
Oxidative stress, often particularly mediated by the reactive oxygen species (ROS) H2O2, has been implicated as a component of a great number of disease states including glaucoma. However, the common method by which oxidative stress is studied in vitro is by bolus dosing, which is not biomimetic in concentration or duration. We recently reported a method for tunable, continuous photocatalytic generation of H2O2 in cell culture that much more closely resembles the oxidative stress profile of chronic diseases such as glaucoma. In this work, we have improved the implementation of the ROS generator and investigated the stability of cultures of primary human trabecular meshwork (HTM) cells under continuous ROS levels over 3 d. We wish to determine a ROS generation rate that would not kill HTM cells.
Photoactive hydrogels were synthesized by mixing a 1:1 volume of poly(ethylene glycol) diacrylate (700 g/mol) with 0.25 mg/mL 2,6-anthraquinone sodium sulfonate. After crosslinking with UV light, hydrogels were punched to 6 mm discs and sterilized with 70% ethanol. HTM cells were plated 50,000/well in a 24-well plate in Hank’s buffered salt solution. Transwell inserts with photoactive hydrogels were placed in each well. An LED emitting 405 nm light was used to illuminate each well of the experimental groups continuously for 3 d. Cells were then fixed and stained with DAPI and nuclei were counted to determine cell numbers. H2O2 was measured by the xylenol orange method.
After 3 d, cells densities were 127 ± 10 and 128 ± 10 cells/mm2 for control and ROS-exposed groups, respectively. The steady-state concentration of H2O2 was 400 nM without cells, which corresponds to a generation rate of 150 nM/min according to the kinetic model.
Following continuous ROS exposure, the HTM cells in the experimental groups had similar cell densities compared to control groups indicating no loss of cells during the culture period. The total dose was 650 µM H2O2 over the duration of the study which would otherwise prove fatal as a bolus. The culture system described here can be used in future studies to examine the cellular response to chronic oxidative loads. This system enables monitoring changes in gene and protein expression levels that provide insights into alterations in the HTM during the progression of glaucoma.
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