(W1030-03-20) Assessing the Impact of Varying DMSO Concentrations on Immunogenicity Readouts in Macrophage and Dendritic Cell-Based In Vitro Models

Purpose: Dimethyl sulfoxide (DMSO) is widely used as a universal solvent for solubilizing drugs, antigens, adjuvants, and other polar compounds in in vitro studies. However, DMSO possesses anti-inflammatory and immunomodulatory properties, which may interfere with immune cell functions. This study aims to determine the safe concentration range of DMSO (% v/v) that can be employed in in vitro immunogenicity assays using immune cell lines, without significantly altering immune response readouts. Nitric oxide (NO) release was measured as a marker of innate immune activation, while interleukin-6 (IL-6) secretion was assessed to understand DMSO's influence on the crosstalk between innate and adaptive immunity. Cell viability was evaluated via MTS assay to establish a cytotoxicity threshold.

Methods: To evaluate the safe in vitro concentration range of DMSO, murine-derived RAW264.7 macrophages and DC2.4 dendritic cells were selected to model innate immune responses. Cells were exposed to a concentration gradient of DMSO: 0.25%, 0.5%, 0.75%, 1.0%, 1.5%, 2.0%, and 2.5% v/v. Lipopolysaccharide (LPS; Thermo Fisher Scientific) was used as a positive control stimulant. Cytotoxicity (MTS assay), nitric oxide (NO) production (Griess reagent assay), and interleukin-6 (IL-6) secretion (ELISA) were measured at 24 and 48 hours to assess time-dependent effects on cell viability and immune activation. Experiments were conducted in triplicate to ensure inter- and intra-day reproducibility. Data were analyzed using one-way ANOVA in GraphPad Prism software to determine statistical significance across treatment groups.

Results: MTS assay results in Figure 1 demonstrated that DMSO concentrations up to 1.0% v/v did not significantly affect the viability of RAW264.7 and DC2.4 cells at both 24 and 48 hours when compared to the untreated control group. However, a noticeable reduction in cell viability was observed at concentrations of 1.5% and above, indicating dose-dependent cytotoxicity beyond the 1% threshold. In the nitric oxide (NO) assay, DC2.4 cells stimulated with 100 ng/mL LPS showed no change in NO release across DMSO concentrations from 0% to 1.5% at 24 hours. However, at higher concentrations (2.0% and 2.5%), NO production was reduced at 48 hours, and a time-dependent effect was observed, with NO levels starting to decline at concentrations above 1.0% DMSO. In contrast, RAW264.7 cells showed reduced NO release at concentrations above 0.25% DMSO at both 24 and 48 hours, which can be seen in Figure 2. These findings suggest that dendritic cells (DC2.4) are more tolerant to DMSO and may serve as a more robust model for NO-based innate immune assays compared to RAW264.7 macrophages. For IL-6 release, DC2.4 cells exposed to LPS and increasing concentrations of DMSO (0% to 2.0%) showed no reduction in IL-6 secretion up to 0.5% DMSO. Beyond this point, IL-6 levels began to decline. Interestingly, RAW264.7 cells maintained stable IL-6 release up to 1.0% DMSO, with reduced levels observed at higher concentrations. Notably, RAW cells secreted approximately 10 times higher levels of IL-6 than DC2.4 cells under similar conditions in Figure 3. This highlights the suitability of RAW264.7 macrophages for IL-6-based immune readouts, whereas DC2.4 cells are preferable for NO-based assays due to their greater DMSO tolerance and stable response profiles.

Conclusion: This study identifies 1.0% v/v as a generally safe upper limit for DMSO use in in vitro immune assays, beyond which cytotoxic and immunomodulatory effects become evident. While both RAW264.7 and DC2.4 cell lines maintained viability up to this threshold, immune readouts showed cell line-specific sensitivities. DC2.4 cells demonstrated greater resilience to DMSO in nitric oxide (NO) release, making them more suitable for assessing innate immune activation under solvent exposure. Conversely, RAW264.7 cells produced significantly higher levels of IL-6 and remained functionally stable up to 1.0% DMSO, suggesting they are better suited for cytokine-based assays involving IL-6. These findings provide practical guidance for selecting DMSO concentrations and immune cell models in in vitro immunogenicity evaluations of antigens, adjuvants, and novel formulations, ensuring data reliability without solvent-induced bias. However, the findings are limited to murine immune cell lines and may not fully translate to human models or other immune parameters, warranting further validation across species and additional cytokine markers.

Acknowledgements: This research was funded by the School of Pharmacy at MCPHS University, Boston.