(170a) Fast, Efficient and Gentle Transfection of Human Adherent Cells in Suspension

The current strategy for delivering genetic materials to adherent cells using gene delivery vehicles (GDVs), involves incubating the GDV solution over a cultured cell monolayer, and its performance is limited by slow mass transport of GDVs to the cell surface. We demonstrate a novel suspension-based transfection method that provides efficient delivery of genetic materials to clinically relevant human cell types, such as induced pluripotent stem cells and fibroblasts, using lipid- and polymeric-based commercial transfection reagents (FugeneHD and Glycofect). Our method reduces the time required for transfection by one full day. The reason behind the success of our method is that suspension transfection leads to faster transport of GDVs to the cell surface. This resulted in maximum GDVs attachment to cell surface within 15 minute of transfection time, with atleast 2-fold higher binding efficiency of GDVs as compared to the plating method. Our method is also compatible with common techniques to improve gene delivery, such as cell cycle synchronization (6-fold increase in gene expression compared to control), facilitating easy adaptability in existing setups. Furthermore, to preserve cell physiology during transfection in suspension, we designed a microfluidic version of our method where the transfection takes place in a flow. In this device, the GDV and cell contact time is below one minute, and we achieved high binding of GDVs to cells (>90% GDV-bound live cells) and significant gene expression in several human cell types with minimal effect due to the device flow rates. The fast, efficient and generally applicable transfection methodologies that we will present can be used for rapid screening of different delivery systems and have significant potential for scale-up and high-throughput cell therapy applications.

Keywords: pluripotent stem cells, fibroblasts, suspension transfection, flow cytometry, microfluidics