Work

Protocol for in vitro transfection of mRNA-encapsulated lipid nanoparticles

Year: 2025

Type: article

Abstract: Lipid nanoparticle (LNP) platforms are promising for mRNA therapeutics; however, while highly effective in vivo , commercial mRNA-LNPs show reduced transfection efficiency in vitro under serum-starved conditions, limiting mechanistic studies and cell engineering applications. Here, we present a new standard for in vitro transfection of mRNA-LNPs, including cell culture preparation, mRNA-LNP treatment in complete media, and mRNA expression level quantification. This protocol improves efficiency and reproducibility in in vitro screening and ensures consistency in the assessment of mRNA-LNPs. • Instruction for transfection of lipid nanoparticles (LNPs) to cells • Steps for cell/LNP preparation, transfection in complete media, and data quantification • Guidance on standardized procedures across multiple cell lines Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Lipid nanoparticle (LNP) platforms are promising for mRNA therapeutics; however, while highly effective in vivo , commercial mRNA-LNPs show reduced transfection efficiency in vitro under serum-starved conditions, limiting mechanistic studies and cell engineering applications. Here, we present a new standard for in vitro transfection of mRNA-LNPs, including cell culture preparation, mRNA-LNP treatment in complete media, and mRNA expression level quantification. This protocol improves efficiency and reproducibility in in vitro screening and ensures consistency in the assessment of mRNA-LNPs.

Source: STAR Protocols

DOI: 10.1016/j.xpro.2025.104159