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Alternatives to Laboratory Animals - ATLA

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Long-term In Vitro Toxicity Models: Comparisons Between a Flow-cell Bioreactor, a Static-cell Bioreactor and Static Cell Cultures

Patricia Pazos, Salvador Fortaner and Pilar Prieto

In vitro long-term toxicity testing is becoming an important issue in the field of toxicology, and there is a need to develop new model systems that mimic human chronic exposure and its effects. The aim of this work was to test two long-term in vitro toxicity systems which are available, a flow-cell bioreactor (Tecnomouse) and a static cell bioreactor system (CELLine CL 6-well), and to compare them with the use of conventional cell culture flasks. A human cell line, Int 407, was exposed to cadmium chloride (CdCl2; for 4 weeks. Cell numbers and cell viabilities were determined by the trypan blue (TB) exclusion assay and from exclusion of propidium iodide (PI) as determined by flow cytometry; and cell viability and metabolic activity were determined by the MTT assay. In addition, total protein determination and cadmium uptake measurements were performed. The results obtained with TB and PI exclusion did not show clear differences in cell viability with increasing CdCl2 concentration. However, in the static cell-culture systems, an increase in MTT reduction was found at low concentrations of CdCl2. Expression of heatshock protein (Hsp27 and Hsp70) increased differently, depending on the CdCl2 concentration applied and the system used. In summary, of the two bioreactors, the CELLine CL 6-well bioreactor was shown to be the more efficient system for performing long-term cytotoxicity studies. It is easy to handle, it permits the assessment of several endpoints, and sufficient replicates can be made available.