Considerations regarding use of solvents in in vitro cell based assays

Abstract

Cell culture systems are widely used for the investigation of in vitro immunomodulatory effects of medicines and natural products. Since many pharmacological relevant compounds are water-insoluble, solvents are frequently used in cell based assays. Although many reports describe the cellular effects of solvents at high concentrations, only a few relate the effects of solvents used at low concentrations. In this report we investigate the interference of three commonly used solvents: Dimethyl sulfoxide (DMSO), ethanol and β-cyclodextrin with five different cell culture systems. The effects of the solvents are investigated in relation to the cellular production of interleukin (IL)-6 or reactive oxygen species (ROS) after lipopolysaccharide (LPS) stimulation. We show that DMSO above 1 % reduces readout parameters in all cell types but more interestingly the 0.25 and 0.5 % solutions induce inhibitory effects in some cell types and stimulatory effects in others. We also found that LPS induced ROS production was more affected than the IL-6 production in the presence of ethanol. Finally we showed that β-cyclodextrin at the investigated concentrations did not have any effect on the LPS induced IL-6 production and only minor effects on the ROS production. We conclude that the effects induced by solvents even at low concentrations are highly relevant for the interpretation of immunomodulatory effects evaluated in cell assays. Furthermore, these results show the importance of keeping solvent concentrations constant in serial dilution of any compound investigated in cell based assays.

Fig. 1

Effects of DMSO. Influence of DMSO concentration (0.25–4 %) on the LPS induced ROS production in HL-60 cells (closed circles) and isolated leukocytes (closed triangles) and on the IL-6 production in isolated PBMCs (open squares), Mono Mac 6 cells (open triangles) and RAW 264.7 cells (open circles). All responses were normalized to the LPS induced responses of the respective cells incubated in HBSS in the absence of DMSO (=100 %). Results are shown as mean ± SEM (n = 3)

Fig. 2

Effects of ethanol. Influence of ethanol concentration (0.01–5 %) on the LPS induced ROS production in HL-60 cells (closed circles) and isolated leukocytes (closed triangles) and on the IL-6 production in isolated PBMCs (open squares), Mono Mac 6 cells (open triangles) and RAW 264.7 cells (open circles). All responses were normalized to the LPS induced responses of the respective cells incubated in HBSS in the absence of DMSO (=100 %). Results are shown as mean ± SEM (n = 3)

Fig. 3

Effects of β-cyclodextrin. Influence of β-cyclodextrin concentration (0.1–100 μg/ml) on the LPS induced ROS production in HL-60 cells (closed circles) and isolated leukocytes (closed triangles) and on the IL-6 production in isolated PBMCs (open squares), Mono Mac 6 cells (open triangles) and RAW 264.7 cells (open circles). All responses were normalized to the LPS induced responses of the respective cells incubated in HBSS in the absence of DMSO (=100 %). Results are shown as mean ± SEM (n = 3)