In situ electroporation of mammalian cells through SiO2 thin film capacitive microelectrodes.

Electroporation is a widely used non-viral technique for the delivery of molecules, including nucleic acids, into cells. Recently, electronic microsystems that miniaturize the electroporation machinery have been developed as a new tool for genetic manipulation of cells in vitro, by integrating metal microelectrodes in the culture substrate and enabling electroporation in-situ. We report that non-faradic SiO2 thin film-insulated microelectrodes can be used for reliable and spatially selective in-situ electroporation of mammalian cells. CHO-K1 and SH-SY5Y cell lines and primary neuronal cultures were electroporated by application of short and low amplitude voltage transients leading to cell electroporation by capacitive currents. We demonstrate reliable delivery of DNA plasmids and exogenous gene expression, accompanied by high spatial selectivity and cell viability, even with differentiated neurons. Finally, we show that SiO2 thin film-insulated microelectrodes support a double and serial transfection of the targeted cells.

Mechanical and electrophysiological properties of the sarcolemma of muscle fibers in two murine models of muscle dystrophy: col6a1-/- and mdx.

This study aimed to analyse the sarcolemma of Col6a1-/- fibers in comparison with wild type and mdx fibers, taken as positive control in view of the known structural and functional alterations of their membranes. Structural and mechanical properties were studied in single muscle fibers prepared from FDB muscle using atomic force microscopy (AFM) and conventional electrophysiological techniques to measure ionic conductance and capacitance. While the sarcolemma topography was preserved in both types of dystrophic fibers, membrane elasticity was significantly reduced in Col6a1-/- and increased in mdx fibers. In the membrane of Col6a1-/- fibers ionic conductance was increased likely due to an increased leakage, whereas capacitance was reduced, and the action potential (ap) depolarization rate was reduced. The picture emerging from experiments on fibers in culture was consistent with that obtained on intact freshly dissected muscle. Mdx fibers in culture showed a reduction of both membrane conductance and capacitance. In contrast, in mdx intact FDB muscle resting conductance was increased while resting potential and ap depolarization rate were reduced, likely indicating the presence of a consistent population of severely altered fibers which disappear during the culture preparation.

Space and time-resolved gene expression experiments on cultured mammalian cells by a single-cell electroporation microarray.

Single-cell experiments represent the next frontier for biochemical and gene expression research. Although bulk-scale methods averaging populations of cells have been traditionally used to investigate cellular behavior, they mask individual cell features and can lead to misleading or insufficient biological results. We report on a single-cell electroporation microarray enabling the transfection of pre-selected individual cells at different sites within the same culture (space-resolved), at arbitrarily chosen time points and even sequentially to the same cells (time-resolved). Delivery of impermeant molecules by single-cell electroporation was first proven to be finely tunable by acting on the electroporation protocol and then optimized for transfection of nucleic acids into Chinese Hamster Ovary (CHO-K1) cells. We focused on DNA oligonucleotides (ODNs), short interfering RNAs (siRNAs), and DNA plasmid vectors, thus providing a versatile and easy-to-use platform for time-resolved gene expression experiments in single mammalian cells.

A potential role for the vanilloid receptor TRPV1 in the therapeutic effect of curcumin in dinitrobenzene sulphonic acid-induced colitis in mice.

A protective role of the transient potential vanilloid receptor 1 (TRPV1) in intestinal inflammation induced by dinitrobenzene sulphonic acid (DNBS) has been recently demonstrated. Curcumin, the major active component of turmeric, is also able to prevent and ameliorate the severity of the damage in DNBS-induced colitis. We evaluated the possibility that curcumin (45 mg kg(-1) day p.o. for 2 days before and 5 days after the induction of colitis) was able to reduce DNBS-induced colitis in mice, by acting as a TRPV1 agonist. Macroscopic damage score, histological damage score and colonic myeloperoxidase (MPO) activity were significantly lower (by 71%, 65% and 73%, respectively; P < 0.01), in animals treated with curcumin compared with untreated animals. Capsazepine (30 mg kg(-1), i.p.), a TRPV1 receptor antagonist, completely abolished the protective effects of curcumin. To extend these data in vitro, Xenopus oocytes expressing rat TRPV1 were examined. Capsaicin-evoked currents (3.3 micromol L(-1)) disappeared subsequent either to removal of the agonist or subsequent to the addition of capsazepine. However, curcumin (30 micromol L(-1)) was ineffective both as regard direct modification of cell membrane currents and as regard interference with capsaicin-mediated effects. As sensitization of the TRPV1 receptor by mediators of inflammation in damaged tissues has been shown previously, our results suggest that in inflamed, but not in normal tissue, curcumin can interact with the TRPV1 receptor to mediate its protective action in DNBS-induced colitis.