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R.D. Braun, A. Beatty, A. Abbas; Oxygen Consumption of Human Choroidal Melanoma Cells in 3-D Culture . Invest. Ophthalmol. Vis. Sci. 2003;44(13):1565.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Local oxygen level is an important determinant of tumor response to different treatments (e.g., radiation therapy) and has been identified as a prognostic factor in several types of cancer. Tumor oxygenation is dependent upon oxygen supply from a chaotic vasculature and oxygen consumption by the tumor cells. In an effort to understand and characterize oxygen consumption (QO2) by human choroidal melanoma parenchyma, we have developed a system to determine oxygenation and QO2 in a 3-D culture of OCM-1 human choroidal melanoma cells. Methods: OCM-1 human choroidal melanoma cells were grown as 3-D layers (multicellular layers or MCL) on collagen-coated culture plate inserts. Each MCL was grown at 37oC in a sealed glass jar containing RPMI continuously exposed to air + 5% CO2. After 10 days, the MCL was removed and suspended in a water-jacketed chamber containing RPMI, bubbled with either air or 5% O2. A recessed-cathode oxygen microelectrode was introduced into the chamber using a motorized micromanipulator, and oxygen tension (PO2) profiles were recorded across the MCL in 20 µm steps. In 6 MCL, profiles were only recorded while the medium was bubbled with air. In 3 other MCL, profiles were recorded on both air and 5% O2. The resultant profiles were fitted to a diffusion model to determine QO2. Results: Thickness of the MCLs ranged from 480-1500 µm. The PO2 profiles were fitted best by either a two-layer model or a modified three-layer model, with each region having a different QO2. The average QO2 on air was 0.380 ± 0.138 ml O2/[100g min] (mean ± SD, n=26), while on 5% O2 it was 0.148 ± 0.095 ml O2/[100g min] (n=8). Air was able to supply enough oxygen to sustain consumption in only 6 of the 26 profiles. Any MCL >800-900 µm in thickness had regions of zero QO2. Interestingly, QO2 of tumor cells near the membrane was higher than QO2 of cells close to the free surface (0.910 ± 0.337 vs. 0.318 ± 0.286 ml O2/[100g min], respectively), even though the PO2 was lower at the membrane (94 ± 10 vs. 148 ± 7 mm Hg, n=26). Conclusions: The model proved to be useful for determining oxygenation and QO2 in 3-D choroidal melanoma cultures under different conditions. The average QO2 of the OCM-1 cells is lower than that of most normal tissues and some other tumors. As expected, bubbling with 5% O2 resulted in lower oxygen levels and lower overall, average QO2 compared to air bubbling. The difference in the QO2 of tumor cells at the two surfaces is either a result of differences in cell density or metabolic activity in the two regions. In the future, this model will permit the study of parameters important in tumor oxygenation in vitro before examining them in animal models.
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