Stretch-based cell stimulation for non-viral gene delivery

Introduction: Non-viral vectors are cationic lipids or polymers that spontaneously assemble with nucleic acids into complexes to drive the exogenous genetic material into cells and alter specific cell functions [1]. Since their first introduction non-viral vectors have made strides forward, but biological barriers that complexes have to overcome still restrict their application in practice [2]. In this context, we propose a new in vitro technology able to boost the transfection efficiency (TE) of the gold standard polymeric vector branched polyethylenimine (bPEI). The novelty of such work is altering cells behaviour through cyclic stretch-based stimulation to ease the cell/complex interactions, and increase the uptake and expression of the transgene. Methods: The stimulation device consists of an electro-mechanical actuator controlled by an Arduino microcontroller able to exert equibiaxial cyclic deformation to silicone cell culture chambers through the vertical displacement of a puncher (nominal deformation (NE): 0-20%; frequency (f): 0-2Hz) (Fig.1A). NE and the strain profile on culture substrates were assessed varying the puncher displacement through a Finite Element Analysis (FEA) using Abaqus®, and validated experimentally. To shed light on the cell response to cyclic stretch, TE of bPEI/pGL3 complexes on stimulated C2C12 cells (stimulation time (t): 30min, NE: 5-10%, f: 1.5Hz) was compared to that of statically-transfected cells. Results: Equibiaxial deformation of culture substrates was confirmed overlapping the experimental NE measurements in 2 orthogonal directions (i.e., NE11 and NE22) at different levels of puncher displacement (Fig.1B). The overlap of experimental and computational NE data assessed the consistency of FEA (Fig.1C). FEA revealed the uniformity of NE distribution in the middle region of culture substrates (corresponding to 1/3 of the total culture chamber) (Fig.1D-E) for NEs ranging from 0-20%. This was used as the cell-seeding area for in vitro transfection experiments. Of note, TE was 4-fold higher in cyclically stretched cells than in unstimulated cells (Fig.1F). Conclusion: We developed a versatile culture system exerting a homogeneous equibiaxial cyclic strain on cell cultures. Overall, the coupling of well-defined mechanical cell stimulation with chemical vectors demonstrated to be promising for driving effective gene transfer. Acknowledgements: We would like to thank Politecnico di Milano for financial support.