P056 - Design of Non-viral Gene Activated Cryogels for Improving the Transfection of Human Mesenchymal Stem Cells.
- N. Carballo-Pedrares (Coruña (A), ES)
- A. Rey Rico (Coruña (A), ES)
- J. Lopez-Seijas (Coruña (A), ES)
- D. Miranda-Balbuena (Coruña (A), ES)
- N. Carballo-Pedrares (Coruña (A), ES)
Abstract
Purpose
Gene-activated matrixes (GAMs) are promising tools for cartilage tissue repair due to their capacity to host selected cell populations, allowing for their transfection within the macroporous structure. In this study, we evaluated the potential of hyaluronic acid (HA)-based cryogels to host human mesenchymal stem cells (hMSCs) enabling their genetic modification by means a new niosomes-based formulation containing a reporter GFP plasmid (pGFP), while maintaining their viability.
Methods and Materials
Hyaluronic acid-based cryogels were synthetized by redox induced free-radical polymerization of MetHA in presence of ammonium persulfate (APS) and N,N,N′,N′-Tetramethylethylenediamine (TEMED). The resulting scaffolds (1 x 6 mm pieces) were characterized in terms of porosity, biodegradation, and shape memory ability. Cytotoxicity of resulting cryogels after the encapsulation of a hMSCs population, was evaluated using a calcein/propidium iodide staining and quantifying the amount of DNA in each sample at 7, 14 and 21 days. Finally, a new niosomes-based formulation complexing pGFP (P80PX nioplexes) was produced and its transfection efficiency of MSCs investigated, before and after being incorporated into the cryogels.
Results
Cryogels showed a macroporous structure (porosity percentage up to 90%) with biodegradation rate of ~50% after 21 days, and shape memory capacity over 99%. The results of the Live/Dead staining assay showed cytotoxicity profiles under 25% in all cases (Fig.1A) with increasing amounts of DNA after 21 days (Fig.1B). Of note, transfection efficiency of hMSCs with P80PX nioplexes was similar to that achieved with the commercial reagent Lipofectamine (Fig.2A) and showed a significantly lower cytotoxicity than this last one, in both monolayer cultures and when encapsulated into the matrixes (Fig.2B).
Conclusion
The developed systems were able to preserve the viability of hMSCs allowing for their proliferation and transfection in a 3D environment. Further studies including therapeutic genes to promote hMSCs chondrogenic differentiation will determine the potential of these matrixes as effective tools for cartilage repair.