Abstract
We describe a method for monitoring cell proliferation in a small-scale hollow-fiber bioreactor (culture volume: 1 ml) by use of the Alamar Blue™ dye. Alamar Blue™ is a non-fluorescent compound, which yields a fluorescent product after reduction, e.g. by living cells. In contrast to the MTT-assay, the Alamar Blue™ assay does not lead to cell death. However, when not removed from the cells, the Alamar Blue™ dye shows a reversible, time- and concentration-dependent growth inhibition as observed for the leukemic cell lines CCRF-CEM, HL-60 and REH. When applied in the medium compartment of a hollow-fiber bioreactor system, the dye is delivered to the cells across the hollow-fiber membrane, reduced by the cells and released from the cell into the medium compartment back again. Thus, fluorescence intensity can be measured in medium samples reflecting growth of the cells in the cell compartment. This procedure offers several advantages. First, exposure of the cells to the dye can be reduced compared to conventional culture in plates. Second, handling steps are minimized since no sample of the cells needs to be taken for readout. Moreover, for the exchange of medium, a centrifugation step can be avoided and the cells can be cultivated further. Third, the method allows discriminating between cell densities of 10 5, 10 6 and 10 7 of proliferating HL-60 cells cultivated in the cell compartment of the bioreactor. Measurement of fluorescence in the medium compartment is more sensitive compared to glucose or lactate measurement for cell counts below 10 6 cells/ml, in particular. We conclude that the Alamar Blue™-assay combined with a hollow-fiber bioreactor offers distinct advantages for the non-invasive monitoring of cell viability and proliferation in a closed system.
Published Version
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