Narrow Results

We report in the present paper an ab initio study on the electronic and structural properties of phospholipidic membranes under the influence of electric fields. We show that the external field alters the charge distribution of the molecule leading to a modification in the electric dipole moment. The torque on the phospholipid may then cause a transmembranar stress, which by its turn, weakens the membrane allowing to the formation of a pore.

The interaction of electric fields with lipid membranes and cells has been extensively studied in the last decades. The phenomena of electroporation and electrofusion are of particular interest because of their widespread use in cell biology and biotechnology. Giant vesicles, being of cell size and convenient for microscopy observations, are the simplest model of the cell membrane. However, optical microscopy observation of effects caused by electric DC pulses on giant vesicles is difficult because of the short duration of the pulse. Recently this difficulty has been overcome in our lab. Using a digital camera with high temporal resolution, we were able to access vesicle fusion dynamics on a sub-millisecond time scale. In this report, we present some observations on electrodeformation and –poration of single vesicles followed by an extensive study on the electrofusion of vesicle couples. Finally, we suggest an attractive approach for creating multidomain vesicles using electrofusion and present some preliminary results on the effect of membrane stiffness on the fusion dynamics.

Electroporation is the formation of reversible pores in cell membranes under a brief pulse of high electric field. Dynamics of pore formation during electroporation suggests that the transmembrane potential would settle approximately at the threshold transmembrane potential and the transmembrane resistance would decrease significantly from the state of relaxation. The current electric circuit equivalent models for electroporation containing time-invariant, static and passive components are unable to capture the pore dynamics. A biophysically-inspired electric circuit equivalent model containing dynamic components for membrane pores has been derived using biological parameters. The model contains a voltage-controlled resistor driven by a two-stage cascaded integrator that is activated through a voltage-gated switch. Simulation results with the derived model showed higher accuracy compared to a commonly used model, where the transmembrane resistance decreased million-fold at the onset of electroporation and the transmembrane potential settled at 99.5% of the critical transmembrane potential, thus enabling improved dynamic behavior modeling ability of the pores in electroporation. The derived model allows fast and reliable analysis of this biophysical phenomenon and potentially aids in optimization of various parameters involved in electroporation.

Electroporation creates transient openings in the cell membrane, allowing for intracellular delivery of diagnostic and therapeutic substances. The degree of cell membrane permeability during electroporation plays a key role in regulating the size of the delivery payload as well as the overall cell viability. A microfluidic platform offers the ability to electroporate single cells with impedance detection of membrane permeabilization in a high-throughput, continuous-flow manner. We have developed a flow-based electroporation microdevice that automatically detects, electroporates, and monitors individual cells for changes in permeability and delivery. We are able to achieve the advantages of electrical monitoring of cell permeabilization, heretofore only achieved with trapped or static cells, while processing the cells in a continuous-flow environment. We demonstrate the analysis of membrane permeabilization on individual cells before and after electroporation in a continuous-flow environment, which dramatically increases throughput. We have confirmed cell membrane permeabilization by electrically measuring the changes in cell impedance from electroporation and by optically measuring the intracellular delivery of a fluorescent probe after systematically varying the electric field strength and duration and correlating the pulse parameters to cell viability. We find a dramatic change in cell impedance and propidium iodide (PI) uptake at a pulse strength threshold of 0.87 kV/cm applied for a duration of 1 ms or longer. The overall cell viability was found to vary in a dose dependent manner with lower viability observed with increasing electric field strength and pulse duration. Cell viability was greater than 83% for all cases except for the most aggressive pulse condition (1kV/cm for 5ms), where the viability dropped to 67.1%. These studies can assist in determining critical permeabilization and molecular delivery parameters while preserving viability.

Pulsed electric field (PEF) is an emerging technology for biomass processing and fractionation by electroporation of cell membrane. Nevertheless, PEF technology and devices require tailoring and adaptation for each specific type of biomass. Such an optimization requires convenient and adaptable laboratory systems, which will enable both electrical and mechanical parameters determination before process upscaling. In this work, we report on the design and development of a laboratory PEF system that allows applying for up to 4kV, 1kA pulses with 1–100$\mu$s and total power dissipation of 20W and up to 25kg of mechanical load. The design of an asymmetric voltage multiplying circuit allows for controlling pulse parameters for each pulse in series. Such an approach enables precise adaptation of PEF to the changing conductivity of the biomass, minimizing the total invested energy in the process. The system was tested on highly conductive marine macroalgae Ulva sp, a promising but challenging feedstock for the biorefinery. This work provides a design of an adaptable PEF device, important for biomass processing with electroporation.

A microelectrode array-based cell electrofusion chip was fabricated by using the MEMS technique. Because of the short distance between two counter microelectrodes, the working voltage on this chip was only 1/100–1/20 as that in the traditional cell electrofusion method. Simulation method was used to analyze the on-chip electric field distribution and optimize the structure of the microelectrodes. The results showed the length and width of the microelectrode, and the distance between two microelectrodes in the horizontal and vertical direction would impact the strength and distribution of the electric field. Thus, optimized chip architecture was obtained, on which six individual chambers were integrated. At least 1680 microelectrodes were patterned within any one chamber. Alternating current signals have been used to manipulate and align cells, and most cells were aligned as cell–cell twins. High-intensity (~10³ V/cm) electric pulses were used to fuse the aligned cell–cell twins. The fusion efficiency was about 40%, which was much higher than that in traditional chemical method (less than 1‰) and electrofusion methods (less than 5%).

To unravel the cellular/molecular mechanisms underlying the development of mammalian brain, we have improved the manipulation techniques of embryos/brain tissues whereby various gene expression vectors are easily transferred into the developing nervous system. These techniques allow us to perform time- and region-specific manipulations of candidate genes and are necessary for a better understanding of the roles of such genes in the developing cerebral cortex.

To produce precise number of neurons and glial cells from neuroepithelial cells, the progeression and exit of the cell cycle should accurately be coordinated. In mammalian neuroepithelial cells, the molecular and cellular mechanisms coordinating the cell cycle progression and neuronal differentiation is yet unknown. Recently, we noticed that a certain cell cycle regulator protein localized in the basal endfeet of the mouse neuroepithelial cells. However, it is difficult to know in which cell cycle phase the protein localizes, because suitable cell cycle markers expressed at the endfeet of the neuroepithelial cells are missing. To address this issue, we performed sequential labeling of neuroepithelial cells by introducing EGFP-F cDNA into cultured mouse embryos using electroporation and short pulse labeling of S-phase cells by incorporating BrdU, and analyzed the cell cycle phase of EGFP-F labeled cells by using antibodies to BrdU and PH3 (a M-phase marker). We found that EGFP-F-labled cells were in S-phase to Gl-phase, but not in G2-phase to M-phase 6 hours after electroporation, and that both of the nucleus and the whole outline of neuroepithelial cell including bipolar processes were clearly visualized. These results suggest that our labeling strategy makes it possible to characterize the cell cycle of neuroepithelial cells, and therefore the cell cycle-dependent distribution of cytoplasmic proteins at the endfeet would be elucidated in the future.

The vertebrate nervous system consists of a huge number of neurons and glial cells. These cell types are generated from neuroepithelial cells at precise positions during development. However, little is known about how kinetics of neuroepithelial cells is regulated, and how the balance of proliferation and differentiation are genetically coordinated. To elucidate cellular mechanisms underlying such complex cell behaviors in the neuroepithelium, we established time-lapse imaging system by applying confocal laser-scanning microscopy for the rat spinal cord slice culture. By electroporation the spinal cord with expression plasmids of fluorescent proteins, nuclear movement of neuroepithelial cells and morphological change on their radial fibers were clearly visualized. Four-dimensional (4-D) time-lapse analyses allowed us to investigate the spatiotemporal dynamics of neuroepithelial cells. These approaches make it possible to analyze functions of genes that regulate cell kinetics and cell morphology.