Nucleic acids have clear clinical potential for gene therapy. Plasmid DNA (pDNA) was the first nucleic acid to be pursued as a therapeutic molecule. Recently, mRNA came into play as it offers improved safety and affordability. In this study, we investigated the uptake mechanisms and efficiencies of genetic material by cells. We focused on three main variables: (1) the nucleic acid (pDNA, or chemically modified mRNA); (2) the delivery vector (Lipofectamine™️3000 or 3DFect™️); and (3) three human primary cells (mesenchymal stem cells, dermal fibroblasts, and osteoblasts). In addition, transfections were studied in a 3D environment using electrospun scaffolds. Cellular internalization of the complexes was assessed by using chemical inhibitors of endocytic pathways. Correlative light and electron microscopy were also used for this purpose. While lipoplexes utilized several entry routes, uptake via caveolae served as the main route for gene delivery. pDNA yielded higher expression levels in fast-dividing fibroblasts, whereas, in slow-dividing osteoblasts, cmRNA was responsible for high protein production. In the case of mesenchymal stem cells, which presented an intermediate doubling time, the combination vector/nucleic acid seemed more relevant than the nucleic acid per se. In all cases, protein expression was higher when the cells were seeded on 3D scaffolds.