Vladimir L. Sukhorukov

Electromanipulation of mammalian cells: fundamentals and application

Electroinjection of membrane-impermeable xenomolecules into freely suspended mammalian cells (so-called electroporation) and cell-to-cell electrofusion are powerful tools for manipulation of the genom and the cytosol of cells. Both field pulse techniques are based on the temporary increase of the membrane permeability due to reversible electrical breakdown of the plasma membrane upon application of external high-intensity field pulses of very short duration. Membrane charging and permabilization caused by high-intensity field pulses are preceded and accompanied by transient electrodeformation forces, which lead to an elongation of the cells in low-conductivity media, thus affecting the membrane area of electropermabilization in response to a breakdown pulse. Transient stretching force assumes a maximum value in low-conductivities pulse media. This facilitates incorporation of membrane-impermeable xenomolecules and field-mediated hybridization as well. Therefore, high and reproducible fields of (genetically) manipulated cells can be expected provided that: 1) the duration of the high-intensity field pulses does not exceed shout 100 /spl mu/s and 2) that the (pulse or fusion) media are hypo-osmolar and exhibit a relatively low conductivities. Such media are also beneficial because field-inducted apoptosis does not occur under these conditions (in contrast to highly conductive media). Indeed, electroporation and electrofusion protocols that fulfill these requirements lead: 1) to high incorporation rates of plasmids (DNA) or artificial chromesomes into living cells without deterioration and 2) to the production of hybridoma cells (by fusion of tumor-infiltrating lymphocytes with heteromyeloma cells), which secrete functional human monoclonal antibodies. Human monoclonal antibodies that bind to and induce apoptosis in autologous tumor cells are promising gents for cancer treatment, as shown by first clinical trials.

Reversible Electropermeabilization of Mammalian Cells by High-Intensity, Ultra-Short Pulses of Submicrosecond Duration

Abstract. Mouse myeloma cells were electropermeabilized by single square-wave electric pulses with amplitudes of up to ∼150 kV/cm and durations of 10–100 nsec. The effects of the field intensity, pulse duration and medium conductivity on cell viability and field-induced uptake of molecules were analyzed by quantitative flow cytometry using the membrane-impermeable fluorescent dye propidium iodide as indicator molecule. Despite the extremely large field strengths, the majority of cells survived the exposure to ultra-short field pulses. The electrically induced dye uptake increased markedly with decreasing conductivity of the suspending medium. We assigned this phenomenon to the transient electrodeformation (stretching) force that assumes its maximum value if cells are suspended in low-conductivity media, i.e., if the external conductivity σe is smaller than that of the cytosol σi. The stretching force vanishes when σe is equal to or larger than σi. Due to their capability of delivering extremely large electric fields, the pulse power systems used here appear to be a promising tool for the electropermeabilization of very small cells and vesicles (including intracellular organelles, liposomes, etc.).

Intracellular Delivery of Trehalose into Mammalian Cells by Electropermeabilization

The disaccharide trehalose is increasingly being used as a very efficient stabilizer of cells, membranes and macromolecules during cryo- and lyoconservation. Although extracellular trehalose can reduce cryo- and lyodamage to mammalian cells, the sugar is required on both sides of the plasma membrane for maximum protection efficiency. In the present study, mouse myeloma cells were loaded with the disaccharide by means of reversible electropermeabilization in isotonic trehalose-substituted medium, which contained 290 mM trehalose as the major solute. By using the membrane-impermeable fluorescent dye propidium iodide as the reporter molecule, optimum electropulsing conditions were found, at which most permeabilized cells survived and recovered (i.e., resealed) their original membrane integrity within a few minutes after electric treatment. Microscopic examination during the resealing phase revealed that electropulsed cells shrank gradually to about 60% of their original volume. The kinetics of the dye uptake and the volumetric response of cells to electropulsing were analyzed using a theoretical model that relates the observed cell volume changes to the solute transport across the transiently permeabilized cell membrane. From the best fit of the model to the experimental data, the intracellular trehalose concentration in electropulsed cells was estimated to be about 100 mM. This loading efficiency compares favorably to other methods currently used for intracellular trehalose delivery. The results presented here point toward application of the electropermeabilization technique for loading cells with membrane-impermeable bioprotectants, with far-reaching implications for cryo- and lyopreservation of rare and valuable mammalian cells and tissues.

A single-shell model for biological cells extended to account for the dielectric anisotropy of the plasma membrane

Abstract For modeling the polarization of biological cells in electric fields and related AC electrokinetical phenomena, such as electrorotation, dielectrophoresis, etc., dielectric isotropy of the plasma membrane and other cellular compartments is usually assumed. However, this traditional assumption is no longer valid in the case of cell membranes containing mobile charges introduced by the adsorbed hydrophobic ions, such as dipicrylamine, tetraphenylborate, etc. Once partitioned into the membrane, the hydrophobic ions can move freely in the plane of the membrane thus increasing the tangential component of membrane conductivity σ mt . On the contrary, only finite charge displacement, i.e. translocation of hydrophobic ions between the two membrane boundaries, can be induced by the field component normal to the plasma membrane plane. This relaxational effect causes a dielectric dispersion (e.g. in the kHz–MHz range) with a marked increase of the radial membrane permittivity e mr at low field frequencies. In this study we extended the single-shell spherical model of cells in order to account for the dielectrically anisotropic plasma membrane. In contrast to the usual approach, where the plasma membrane permittivity and conductivity are viewed as scalar quantities, these membrane parameters are treated as tensors in the anisotropic membrane model. Calculations based on the new model showed that the tangential conductivity of hydrophobic ions can induce noticeable changes in the low-frequency part of the electrokinetic spectra of cells.

Effects on capacitance by overexpression of membrane proteins

Functional Channelrhodopsin-2 (ChR2) overexpression of about 10(4)channels/mum(2) in the plasma membrane of HEK293 cells was studied by patch-clamp and freeze-fracture electron microscopy. Simultaneous electrorotation measurements revealed that ChR2 expression was accompanied by a marked increase of the area-specific membrane capacitance (C(m)). The C(m) increase apparently resulted partly from an enlargement of the size and/or number of microvilli. This is suggested by a relatively large C(m) of 1.15+/-0.08 microF/cm(2) in ChR2-expressing cells measured under isotonic conditions. This value was much higher than that of the control HEK293 cells (0.79+/-0.02 microF/cm(2)). However, even after complete loss of microvilli under strong hypoosmolar conditions (100 mOsm), the ChR2-expressing cells still exhibited a significantly larger C(m) (0.85+/-0.07 microF/cm(2)) as compared to non-expressing control cells (0.70+/-0.03 microF/cm(2)). Therefore, a second mechanism of capacitance increase may involve changes in the membrane permittivity and/or thickness due to the embedded ChR2 proteins.

Surviving high-intensity field pulses : Strategies for improving robustness and performance of electrotransfection and electrofusion

Electrotransfection and electrofusion, both widely used in research and medical applications, still have to face a range of problems, including the existence of electroporation-resistant cell types, cell mortality and also great batch-to-batch variations of the transfection and fusion yields. In the present study, a systematic analysis of the parameters critical for the efficiency and robustness of electromanipulation protocols was performed on five mammalian cell types. Factors examined included the sugar composition of hypotonic pulse media (trehalose, sorbitol or inositol), the kinetics of cell volume changes prior to electropulsing, as well as the growth medium additives used for post-pulse cell cultivation. Whereas the disaccharide trehalose generally allowed regulatory volume decrease (RVD), the monomeric sugar alcohols sorbitol and inositol inhibited RVD or even induced secondary swelling. The different volume responses could be explained by the sugar selectivity of volume-sensitive channels (VSC) in the plasma membrane of all tested cell types. Based on the volumetric data, highest transfection and fusion yields were mostly achieved when the target cells were exposed to hypotonicity for about 2 min prior to electropulsing. Longer hypotonic treatment (10–20 min) decreased the yields of viable transfected and hybrid cells due to (1) the cell size reduction upon RVD (trehalose) or (2) the excessive losses of cytosolic electrolytes through VSC (inositol/sorbitol). Doping the plasma membrane with lipophilic anions prevented both cell shrinkage and ion losses (probably due to VSC inhibition), which in turn resulted in increased transfection and fusion efficiencies.

Physicochemical features of ultra-high viscosity alginates.

The physicochemical characteristics of the ultra-high viscosity and highly biocompatible alginates extracted from Lessonia nigrescens (UHV(N)) and Lessonia trabeculata (UHV(T)) were analyzed. Fluorescence and (1)H NMR spectroscopies, viscometry, and multi-angle light scattering (MALS) were used for elucidation of the chemical structure, molar mass, and coil size. The sequential structures from NMR spectroscopy showed high guluronate content for UHV(T), but low for UHV(N). Intrinsic viscosity [eta] measurements exhibited unusual high values (up to 2750 mL/g), whereas [eta] of a commercial alginate was only about 970 mL/g. MALS batch measurements of the UHV-alginates yielded ultra-high values of the weight average molar mass (M(w) up to 1.1x10(6) g/mol) and of the z-average gyration radius (R(G)(z) up to 191 nm). The M(w) and R(G)(z) distributions of UHV-alginates and of ultrasonically degraded fractions were determined using size exclusion chromatography combined with MALS and asymmetrical flow-field-flow fractionation. The M(w) dependency of [eta] and R(G)(z) could be described by [eta]=0.059xM(w)(0.78) and R(G)(z)=0.103xM(w)(x). (UHV(N): x=0.52; UHV(T): x=0.53) indicating that the monomer composition has no effect on coil expansion. Therefore, the equations can be used to calculate M(w) and R(G)(z) values of UHV(T)- and UHV(N)-alginate mixtures as used in immunoisolation. Furthermore, the simple and inexpensive capillary viscometry can be used for real-time validation of the extraction and purification process of the UHV-alginates.

ELECTROROTATION OF ERYTHROCYTES TREATED WITH DIPICRYLAMINE : MOBILE CHARGES WITHIN THE MEMBRANE SHOW THEIR SIGNATURE IN ROTATIONAL SPECTRA

Abstract. In this study, electrorotation spectra of individual cells (that is, frequency dependence of cell rotation speed) have been proved to yield information not only about the passive electric properties of cell constituents, but also about the presence of mobile charges within the plasma membrane being part of ion carrier transport systems. Experiments on human erythrocytes pretreated with the lipophilic anion dipicrylamine (DPA) gave convincing evidence that these artificial mobile charges adsorbed to the plasma membrane contributed significantly to the rotational spectrum at relatively low conductivity of the external medium (2–5 mS m−1). Theoretical integration of the mobile charge concept into the single-shell model (viewing the cell as a homogenous sphere surrounded by a membrane) led to a set of equations which predicted electrorotational behavior of DPA-treated cells in dependence on medium conductivity. The quantitative data on the partition and the transmembrane translocation rate of the DPA anion extracted from the experimental rotational spectra agreed well with the corresponding literature values.

Volume regulation of murine T lymphocytes relies on voltage-dependent and two-pore domain potassium channels

A variety of ion channels are supposed to orchestrate the homoeostatic volume regulation in T lymphocytes. However, the relative contribution of different potassium channels to the osmotic volume regulation and in particular to the regulatory volume decrease (RVD) in T cells is far from clear. This study explores a putative role of the newly identified K(2P) channels (TASK1, TASK2, TASK3 and TRESK) along with the voltage-gated potassium channel K(V)1.3 and the calcium-activated potassium channel K(Ca)3.1 in the RVD of murine T lymphocytes, using genetic and pharmacological approaches. K(2P) channel knockouts exerted profound effects on the osmotic properties of murine T lymphocytes, as revealed by reduced water and RVD-related solute permeabilities. Moreover, both genetic and pharmacological data proved a key role of K(V)1.3 and TASK2 channels in the RVD of murine T cells exposed to hypotonic saline. Our experiments demonstrate a leading role of potassium channels in the osmoregulation of T lymphocytes under different conditions. In summary, the present study sheds new light on the complex and partially redundant network of potassium channels involved in the basic physiological process of the cellular volume homeostasis and extends the repertoire of potassium channels by the family of K(2P) channels.

Dielectrophoretic behavior of in vitro-derived bovine metaphase II oocytes and zygotes and its relation to in vitro embryonic developmental competence and mRNA expression pattern.

Selecting developmentally competent oocytes and zygotes based on their morphology is more often influenced by personal judgments and lacks universal standards. Therefore, this experiment was conducted to investigate the rate of development and mRNA level of dielectrophoretically separated oocytes and zygotes to validate dielectrophoresis (DEP) as non-invasive option for selection of oocytes and zygotes. In the first experiment, metaphase II oocytes with (PB(+)) and without (PB(-)) first polar body and zygotes were subjected to DEP at 4 MHz and 450 mum electrode distance and classified into fast, very fast, slow, and very slow depending on the time elapsed to reach one of the electrodes in the electric field. Parthenogenetic activation was employed to monitor the embryonic development of dielectrophoretically classified oocytes. The result revealed that at 6 and 7 days of post-activation, the blastocyst rate of very slow dielectrophoretic PB(+) and PB(-) oocytes was significantly (P < 0.05) lower than other groups. Similarly, in zygotes, the blastocyst rate at 7 days post-insemination was higher (P < 0.05) in the very fast dielectrophoretic categories when compared with the slow and very slow categories. In the second experiment, mRNA level was analyzed in the very fast and very slow dielectrophoretic PB(+) oocytes and zygotes respectively using the bovine cDNA microarray. The result showed that 36 and 42 transcripts were differentially regulated between the very fast and very slow dielectrophoretic categories PB(+) oocytes and zygotes respectively. In conclusion, dielectrophoretically separated oocytes and zygotes showed difference in the rate of blastocyst development accompanied by difference in transcriptional abundances.