Effective Gene Delivery to Mesenchymal Stem Cells Based on the Reverse Transfection and Three-Dimensional Cell Culture System

To enhance the level and prolong the duration of gene expression for gene-engineered rat mesenchymal stem cells (MSCs) using non-viral vector. A novel transfection system based on reverse transfection method and three-dimensional (3D) scaffold was developed. The reverse gene transfection system was evaluated for transfection efficiency compared to conventional methods. Collagen sponge and polyethylene terephthalate non-woven fabric were introduced as scaffolds to perform 3D culture with reverse transfection. pDNA coding TGF beta-1 was delivered to MSCs to assess its ability in inducing chondrogenesis with the 3D non-viral reverse transfection system. The reverse transfection method induced higher transgene levels than the conventional transfection in the presence of serum. The electric charge of the anionic gelatin plays an important role in this system by affecting the release pattern of the gene complexes and through the adsorption of serum protein to the substrate. During a long-time in vitro culture, MSCs cultured on 3D scaffolds exhibited a higher transgene expression level and more sustained transgene expression than those cultured and transfected on the two-dimensional substrate. The combination of reverse transfection system with 3D cell culture scaffold benefits the cell proliferation and long-time gene transfection of MSCs.