Impact of cell line and reporter gene selection on in-vitro transfection evaluation of mRNA lipid nanoparticles

Abstract The mRNA lipid nanoparticle (mRNA-LNP) is a promising platform for vaccines and a variety of therapeutic areas, as demonstrated by the effective mRNA-LNP formulations in COVID-19 vaccines. While in-vitro transfection studies are crucial for optimizing mRNA-LNP formulations, few studies examine how cell line selection and reporter genes affect transfection efficiency. In our study, we investigated the in-vitro transfection efficiency of firefly luciferase mRNA-LNP on Jurkat cells, L-929 cells, and HEK 293 T cells. Jurkat cells, as a suspension cell line, displayed low transfection efficiency. The luciferase expression showed a non-linear relationship with mRNA dose, and cytotoxicity was observed with even low concentrations of mRNA. L-929 cells showed a linear relationship between bioluminescence and mRNA concentration, but only at low levels of mRNA, and their luciferase expression is limited. HEK 293 T cells are superior because of a strong linear dose–response and higher signal intensity. However, when using the luciferase-based assay for mRNA-LNP transfection, we observed high intra-group variations with signal fluctuated among technical replicates of the same formulation. In contrast, eGFP mRNA exhibited high reproducibility for the in-vitro transfection tests (coefficient of variation < 10%) and maintained strong linear correlation between mRNA concentration and eGFP expression (R 2 > 0.95). This study highlights the importance of selecting a cell line model, a reporter gene, and analytical methods for developing a robust and reproducible in-vitro transfection assay for the development of mRNA-LNP formulation.