Comparative in vitro stimulation with lipopolysaccharide to study TNFα gene expression in fresh whole blood, fresh and frozen peripheral blood mononuclear cells

In vitro stimulation with fresh and frozen peripheral blood mononuclear cells (PBMCs) or fresh whole blood has been widely used in animal studies and clinical trials to study the immunological features of a number of human diseases. The objective of this study was to determine the difference in response to stimulation of fresh PBMCs, frozen PBMCs, and fresh whole blood by change of TNFα gene expression levels after 4-hour in vitro lipopolysaccharide (LPS) stimulation. Our results demonstrate that TNFα gene expression significantly increases in both fresh PBMCs and fresh whole blood when 1 μg/ml or 10 μg/ml LPS was used but only after stimulation with 10 μg/ml LPS in frozen PBMCs. Mean fold change of TNFα gene expression levels after 1 μg/ml LPS stimulation was significantly higher using fresh whole blood (51.01 ± 7.91) as compared to fresh PBMCs (8.92 ± 2.16) or frozen PBMCs (3.04 ± 0.55) (p ≤ 0.007). The same trend was also observed with 10 μg/ml LPS stimulation (p ≤ 0.001). Decreased PBMC cell viability due to cryopreservation, cytokine depletion and removal of some minor cells during cell isolation could attribute to the low TNFα expression in the frozen PBMCs. Our results strongly suggest that fresh whole blood is the best choice for in vitro stimulation studies.