A Versatile Platform for Cell Therapy Cytotoxicity Assays Using mRNA Transfection

Código PS612

Abstract

Richard Somberg, Brock Binkowski, Julia Gilden, Nicholas Hess, Aileen Paguio, Mei Cong & Marjeta Urh
Promega Corporation, 2800 Woods Hollow Road, Madison WI 53711

The HiBiT Target Cell Killing Bioassay (HiBiT TCK Bioassay) is a sensitive and selective method for quantifying target cell lysis in coculture experiments with effector cells. In this assay, target cells engineered to express a HiBiT-tagged protein are cocultured with effector cells, where target cell death promotes release of the HiBiT- tagged protein into the extracellular medium. Once there, HiBiT can bind to cell- impermeable LgBiT to generate a bright, luminescent enzyme in the presence of furimazine substrate. In this manner, a non-lytic detection reagent containing LgBiT and furimazine substrate can be used to quantify target cell death in a gain-of-signal assay format with a cumulative readout. To date, this approach has been used exclusively with stable expression of the HiBiT-tagged protein, requiring time and effort to make stable clones.

To expand beyond the use of stable clones, we developed a novel mRNA transfection reagent that enables rapid and uniform expression of HaloTag-HiBiT in a wide range of target cell types. This approach dramatically reduces the workload for creating HiBiT target cells, allowing users to readily label target cell type(s) of interest. We demonstrate labeling of a variety of target cell types and their use in the HiBiT TCK Bioassay. Additionally, we demonstrate the use of mRNA transfection to achieve uniform and highly tunable expression of target receptors in cells stably expressing a HiBiT-tagged protein, allowing drug potency/efficacy testing across a broad range of receptor expression levels in a single cell type.