343. Non-Viral Transfection of Primary Human Fibroblasts in Suspension Microcarrier Culture

Abstract

Suspension bioreactors have been employed for the large-scale production of recombinant biomacromolecules and expansion of primary tissue-derived cells for clinical applications. However, the bioprocess requires establishing a stably-expressing cell line in static culture first, which is a lengthy process that can add significant upfront cost and limit economies of scale. A transient-expression system is a simpler cost-effective platform for rapid production in a small-medium format, which could make it feasible for generating patient-specific cell-based products. However, current methods for the efficient transfection of primary cells involve either physical methods of delivery that are not adaptable for suspension culture, or viral vectors that are potentially mutagenic, which can present an even greater problem in suspension culture that tend to select for high proliferative tumorigenic clones. Thus, a method to efficiently transfect clinically-relevant primary cells directly in suspension culture by non-viral means is needed to streamline the derivation, expansion and production of cell-based product in one integrated bioprocess. In this study, we explored the feasibility of transfecting primary tissue-derived fibroblasts directly in microcarrier suspension culture using non-viral cationic reagents. We first developed an optimized non-viral transfection system by adopting the gWiz High Expression plasmid, which is capable of 5x higher expression than CAG-based episomal plasmids. We further evaluated several commercial cationic reagents for transfection of primary human fibroblast in static culture and found that XtremeGENE HP and TransIT-3D were among the most efficient transfection reagents (up to 60%). Next, in order to transfect anchorage-dependent cells in suspension, we evaluated a number of microcarriers for their suitability in culturing fibroblast in suspension. We focused on examining cell attachment efficiencies and growth rates on the microcarriers since these variables have the most significant impact on the overall transfection efficiencies. While all of the microcarriers surveyed in this study (i.e. polyGEMs with FACTIII, Collagen, Pronectin F, Glass, Plastic, or Plastic+, and CultiSphere S, Cytodex 3, Hillex II) were capable of supporting cell attachment, Cultisphere S and Hillex II had the most conducive surface for attachment (40% and 25% higher compared to tissue culture dishes). These differences in attachment efficiencies translated into different lag phase and exponential phase among the carriers when we subsequently assayed for growth rate on the carriers over a 12-day period using a MTT-based assay. Due to the differences in coating, charged surfaces, and growth rate on these microcarriers, in order to properly assess for their compatibility with cationic transfection reagent in facilitating transfection, we transfected cells at multiple time points (day 2, 3, 5 and 7 days post cell seeding) and found that transfection efficiencies correlated with the time frame in which cells were growing the fastest (Days 2-3); highest efficiencies were seen in cells cultured on Glass, Cytodex 3, CultiSphere S and Hillex II (~12-16%). In summary, we demonstrated here a first step towards the efficient transfection of primary tissue-derived human fibroblasts directly in a suspension microcarrier culture using cationic reagent; additional optimization is expected to bring the efficiencies comparable to those in static culture.