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RESEARCH PRODUCT

An in vitro model to study cellular photosensitizer uptake and photodynamic dose-response relationships of tumor cells

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Cellular fluorescence intensity (CFI) after incubation with varying concentrations of the photosensitizer Photofrin and the photodynamically induced dose-response relationships of hamster melanoma cells (A-MEL-3) were studied in a recently developed in vitro model. After administration of Photofrin to the extracellular serum-free medium, CFI was evaluated by flow cytometry together with constantly fluorescing latex particles used as a reference. After 5 min, 50% of maximal CFI was found, and after 60 min CFI was maximal. No further increase was obtained during the exposure to Photofrin over the incubation period of 4 h. During this plateau phase, CFI was significantly related to the concentration of Photofrin in the extracellular medium (r = 0.94; P0.001). Subsequent to increasing intervals of Photofrin exposure, cells were irradiated with laser light at 630 nm (40 mW/cm2, 4J). Cell viability as evaluated by trypan blue exclusion was significantly decreased with increasing concentrations of Photofrin in the medium, and significantly correlated with CFI during the plateau phase. After photodynamic treatment (PDT) cell fluorescence was reduced by about 15%. This was neither dose- nor time-dependent. On the basis of these findings we propose that CFI indicates photosensitizer uptake. This is also supported by the relation between CFI and phototoxicity. The latter also suggests that CFI might be useful to predict the PDT in vivo efficacy by this in vitro model. Besides measurements of photosensitizer uptake and cell photoxicity, the model demonstrates an excellent opportunity to study the molecular mechanisms of action associated with PDT.