(160b) The Effect of Centrifugal Force on the Mechanism of Wharton’s Jelly Mesenchymal Stem Cells Transfection
In gene therapy, viral vectors such as retrovirus, lentivirus and adenovirus, have been used for the delivery of genes into cells via a process known as transduction. Even though it offers high gene delivery (nearly 99%) and stable gene expression, many limitations remain associated with viral vectors such as insertional mutagenesis, immunogenicity, limited DNA packaging capacity and cumbersome large-scale production. Non-viral gene carriers are promising alternatives for gene delivery and have the potential to address these limitations. Moreover, the transient expression associated with these systems can be more compatible with the natural wound healing processes. However, transfection has significantly lower gene entry rates compared to the viral transduction (approximately 10% to 30% in hMSC). This lower efficiency is due to the mechanism of transfection, which has been theorized to be affected by multiple factors regarding nanoparticle properties, including: size, charge, degradation, endosomal escape and arrival to the nucleus. These barriers depend directly on the chemical nature of the material that covers the gene and its non-specific interaction with all the pathways within the cell. Therefore, there is still need for more in-depth studies, with the aim of improving transfection techniques using this type of vectors or physical mechanisms.
One of the most promising non-viral gene carriers is polyethylenimine (PEI). This polymer, which has a great variety of molecular weights and degrees of branching, has been extensively studied in vitro ever since its initial use as gene carrier. PEI has an advantage over other polycationic polymers in that it acquires a remarkable DNA condensation capability and a potential ability to protect DNA from enzymatic degradation. In addition, the three different types of amines that it has are responsible for generating the buffering capacity over a wide range of pH values. This facilitates endosomal escape of carriers, which has been named the "proton-sponge" effect.
As discussed before, transfection presents a poor efficiency of gene uptake. However, we tested here the hypothesis that the efficiency of gene transfection is limited by a simple physical barrier: low concentration of gene (DNA) on the surface of the cell. Therefore, the present study evaluated the mechanism of centrifugation during the transfection process in order to increase the concentration of DNA on the cell surface, thus generating higher transfection efficiencies. The present study evaluated the mechanism of centrifugation during the transfection process in order to increase the concentration of DNA on the cell surface, which allowed higher transfection efficiencies. For this purpose, mesenchymal stem cells derived from umbilical cord, reporter genes such as GFP and luciferase and, branched PEI 25kDa were used to evaluate and optimize the transfection technique by assessing various centrifugal forces. We were able to standardize a transfection protocol in which the weight ratio and different concentrations of complex were evaluated to obtain transfection rates above 20% with a non-viral method.