Spheroid-based cellular screening approaches represent a highly physiologic experimental setup to identify novel anticancer drugs and an innovative preclinical model to reduce the high failure rate of anticancer compounds in clinical trials. The resazurin reduction (RR) assay, known as the alamarBlue or CellTiter-Blue assay, is frequently used to determine cell viability/ proliferation capacity in eukaryotic cells. Whether this assay is applicable to assess viability in multicellular spheroids has not been evaluated. We analyzed the RR assay to measure cytotoxic and/or cytostatic responses in tumor cell spheroids compared with conventional 2D cultures. We found that tight cell-cell interactions in compact spheroids hamper resazurin uptake and its subsequent reduction to resorufin, leading to lowered reduction activity in relation to the actual cellular health/cell number. Treatment with staurosporine disrupted close cell-cell contacts, which increased resazurin reduction compared with untreated controls. Loss of tight junctions by trypsinization or addition of EGTA or EDTA restored high resazurin reduction rates in untreated spheroids. In conclusion, the RR assay is unsuited to quantitatively measure cellular health/cell number in compact spheroids. However, it can be used to distinguish between cytotoxic versus cytostatic compounds in spheroids. Restoration of the correlation of cell viability/number to resazurin reduction capacity can be achieved by disruption of tight junctions.

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Background Tumor necrosis factor alpha (TNF-α), as a key mediator, represents a major point of attack in sepsis. Since it has been shown that systemic anti-TNF-α antibodies do not improve the situation of septic patients, the use of specific adsorption technology in the treatment of sepsis could have beneficial effects. Methods Magnetic beads coated with polyclonal or with monoclonal anti-TNF-α antibodies were investigated in vitro in order to analyze their ability to prevent TNF-α induced adhesion of peripheral blood mononuclear cells (PBMCs) at human umbilical vein endothelial cells (HUVECs). Additionally, therapeutical monoclonal anti-TNF-α antibodies were proofed for inhibitory effects of TNF-α mediated activation of HUVECs. Results We have shown, in vitro, that beads coated with polyclonal or monoclonal anti-TNF-α antibodies were able to significantly reduce monocyte adhesion. It was possible to decrease monocyte adhesion from nearly 9% to 3% within 2 hours and from 18% to 2% within 6 hours of TNF-α treatment by the simultaneous use of beads coated with polyclonal anti-TNF-α antibodies. Beads coated with monoclonal anti-TNF-α antibodies could even prevent monocyte adhesion within the first 2 hours, and reduced monocyte adhesion to 2% during 6 hours of incubation with TNF-α. On the other hand, application of therapeutic anti-TNF-α antibodies showed no significant difference compared to the measured monocyte adhesion values of activated endothelial cells. Conclusion Adsorption techniques using specific adsorbents, possibly used in MDS (Microspheres-Based Detoxification System), are efficient in specific reduction of TNF-α and pathophysiological consequences, since monocyte adhesion at activated HUVECs was shown to be reduced. (Int J Artif Organs 2006; 29: 1140–7)

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