Monitoring of cell viability and cell growth in a hollow-fiber bioreactor by use of the dye Alamar Blue (TM)

We describe a method for monitoring cell proliferation in a small-scale hol low-fiber bioreactor (culture volume: 1 mi) by use of the Alamar Blue (TM) dye. Alamar Blue (TM) is a non-fluorescent compound, which yields a fluores cent product after reduction, e.g. by living cells. In contrast to the MTT- assay, the Alamar Blue (TM) assay does not lead to cell death. However, whe n not removed from the cells, the Alamar Blue (TM) dye shows a reversible, time- and concentration-dependent growth inhibition as observed for the leu kemic cell lines CCRF-CEM, HL-60 and REH. When applied in the medium compar tment of a hollow-fiber bioreactor system, the dye is delivered to the cell s across the hollow-fiber membrane, reduced by the cells and released from the cell into the medium compartment back again. Thus, fluorescence intensi ty 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 call be reduced compared to conventional culture i n 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 cen trifugation step can be avoided and the cells can be cultivated further. Th ird, 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 i s mon sensitive compared to glucose or lactate measurement for cell counts below 10(6) cells/ml, in particular. We conclude that the Alamar Blue (TM) -assay combined with a hollow-fiber bioreactor offers distinct advantages f or the nan-invasive monitoring of cell viability and proliferation in a clo sed system. (C) 2001 Published by Elsevier Science B.V.