(Invited) Finite Element Model Simulations to Assist the Design of Microdevices Dedicated to the Localized Electroporation of Mouse Embryos

Monday, 6 October 2014: 10:30
Expo Center, 1st Floor, Universal 10 (Moon Palace Resort)
X. Zhao, E. Mazari (CNRS), D. Suárez-Boomgaard, I. Migeotte (Université Libre de Bruxelles), A. Perea-Gomez (Université Paris Diderot), and C. Gosse (CNRS)
During embryonic development interactions between cell groups enable the growth, migration, and specification of various tissues. The ability to follow and modify cell behavior with accurate spatiotemporal resolution thus appears as a prerequisite to study morphogenesis. More precisely, the dynamics of the above mentioned phenomena can be unraveled by manipulating gene expression locally. Electroporation, the delivery of exogenous molecules into targeted cell populations through electric permeation of the plasma membrane, has for instance been harnessed to this aim. However, current strategies suffer from insufficient reproducibility and mediocre survival when applied to early post-implantation mouse embryos. Indeed, between 5.5 and 7.5 days of development theses organisms are small (between 150 and 500 um long) and delicate. Consequently, they require dedicated tools.

Sharp metallic electrodes are efficient to concentrate electric field lines and permeate the plasma membrane over a restricted area. Nevertheless, to do so they have to be very close to the targeted tissue. Since harmful species such as gases and protons are produced when voltage pulses are applied, using the preceding microneedles often results in embryo death. Thereby, we introduce here a new type of device to achieve localized electroporation with high efficiency and reduced cell damage. In our “electrodeless” approach the electric field is generated by remote gold pads and channeled towards the cells to be transfected by dielectric tunnels. Finite element model simulations relying on a simple electrical model of the mouse embryo indicated that the present dielectric guide-based design even perform better than the existing alternatives.

Such a microsystem was next fabricated on a glass wafer, by patterning the guide walls in SU-8 photoresist, hydrophilizing them with an air plasma, and closing the structure with a Parafilm ceiling. Performances were next tested by targeting the distal visceral endoderm (DVE), a migrating cell population essential for anterior-posterior axis establishment. More precisely, penetration of a fluorescent dextran enabled to assert membrane permeation, expression of plasmid coding for a fluorescent protein reported on transfection success, and cell death was evaluated thanks to propidium iodide staining. The size of the dielectric guide aperture as well as the voltage pulse sequence could therefore be optimized and transfection could efficiently and reproducibly be restricted to less than four visceral endoderm cells without compromising cell behavior and embryo survival.

To demonstrate the validity of our approach with respect to biological questions, we combined targeted mosaic expression of fluorescent markers with live imaging in transgenic embryos to reveal new facts concerning tissue rearrangement between 5.5 and 6.5 days of development. For instance, we could observe that, like the leading DVE cells at the migration front, non-leading ones also send long basal projections and intercalate during their displacement. Thus, they are not passively dragged but they participate actively to the collective motion.

Finally, we showed that the use of our microsystem can be extended to a variety of embryological contexts, from preimplantation embryos to organ explants dissected from latter stage organisms. Hence, we have experimentally validated an approach delivering a tailor-made tool for the study of morphogenesis in mouse. Furthermore, we have delineated a comprehensive strategy for the development of ad hoc electroporation devices. Our future work is now dedicated to the fabrication of reusable and robust all-in-glass chip that any embryologist will be able to utilize in his laboratory.