Lentiviral vectors are beginning to emerge as a viable choice for human gene therapy. Here, we describe a method that combines the convenience of a suspension cell line with a scalable, nonchemically based, and GMPcompliant transfection technique known as flow electroporation (EP). Flow EP parameters for serum-free adapted HEK293FT cells were optimized to limit toxicity and maximize titers. Using a third generation, HIVbased, lentiviral vector system pseudotyped with the vesicular stomatitis glycoprotein envelope, both small-and large-volume transfections produced titers over 1 · 10 8 infectious units/mL. Therefore, an excellent option for implementing large-scale, clinical lentiviral productions is flow EP of suspension cell lines.

Electroporation generally refers to the technique of permeabilizing cell membranes by applying a short and intense electric pulse across a cell, such that the barrier function of the membrane is instantaneously compromised. During such time, genetic materials may travel across the membrane. For a successful gene transfer process, the barrier function of the cell membrane is rapidly restored, and the cell survives. The electrotransfection process thus comprises two steps. The first step is electroporation, which is governed by the electrical properties of the cell and the suspension medium. The controlling parameters are mainly electrical. The second step is recovery, which must take into account the biological characteristics of the cells. We consider these two steps in this chapter.