Corning® CellSTACK® Culture Chamber PEIpro® Transfection Protocol

This document provides a generalized procedure for chemical-based DNA transfection using PEIpro transfection reagent in Corning CellSTACK culture chambers. PEIpro transfection reagent is a leading chemically defined linear \polyethylenimine optimized for large-scale virus production in common mammalian producer cell lines¹. The transfection protocol described is intended as a starting point for those wishing to transfect large numbers of cells in the Corning CellSTACK-10 chamber, especially for viral production.

As a simple proof-of-concept, the protocol was developed for HEK293T cells using a high-expression green fluorescent protein (GFP) control plasmid, gWIZ™-GFP (Aldevron Cat. Nos. 5006 or 5007). In this instance, GFP provided a convenient reporter for both visual inspection of the transfected chambers and assay of transfection efficiency using the NucleoCounter® NC-3000™ Advanced Image Cytometer2. The procedure discussed can be adapted for other cell types and to accommodate co-transfection of multiple plasmids for viral vector production. In addition, transfections using PEIpro transfection reagent can be easily scaled for  process optimization in any size Corning CellSTACK culture chamber prior to large-scale transfections in multiple CellSTACK-10 chambers.