Generation of Recombinant Primary Human B Lymphocytes Using Non-Viral Vectors

Abstract

Although the development of gene delivery systems based on non-viral vectors is advancing, it remains a challenge to deliver plasmid DNA into human blood cells. The current “gold standard”, namely linear polyethyleneimine (l-PEI 25 kDa), in particular, is unable to produce transgene expression levels >5% in primary human B lymphocytes. Here, it is demonstrated that a well-defined 24-armed poly(2-dimethylamino) ethyl methacrylate (PDMAEMA, 755 kDa) nano-star is able to reproducibly elicit high transgene expression (40%) at sufficient residual viability (69%) in primary human B cells derived from tonsillar tissue. Moreover, our results indicate that the length of the mitogenic stimulation prior to transfection is an important parameter that must be established during the development of the transfection protocol. In our hands, four days of stimulation with rhCD40L post-thawing led to the best transfection results in terms of TE and cell survival. Most importantly, our data argue for an impact of the B cell subsets on the transfection outcomes, underlining that the complexity and heterogeneity of a given B cell population pre- and post-transfection is a critical parameter to consider in the multiparametric approach required for the implementation of the transfection protocol.

mRNA Sonotransfection of Tumors with Polymeric Microbubbles: Co‐Formulation versus Co‐Administration

“…[ 30 ] However, it is noteworthy that even at the extreme condition of 400 kPa and 60 s sonication time, cell death never exceeded 26.4 ± 3.6 %, which is a number that was still considered acceptable in other gene therapy approaches. [ 31 ]…”

Combined approach of selective and accelerated cloning for microfluidic chip‐based system increases clone specific productivity

“…Transfection methods, concentration of the selection reagent and vector concentration of recombinant protein can impact the pool viability and recovery. [17,18] In our setup, the viability of the pool ranged from 50.4% to 70.6% at WD11, measured with ViCell. Figure 2A represents the viability curve of mAb1.…”