The Effect of Encapsulation in Red Blood Cells on the Distribution of Methotrexate in Mice

Peer Reviewe

Transcellular Ion Flow in Escherichia coli B and Electrical Sizing of Bacterias

Dielectric breakdown of cell membranes and, in response, transcellular ion flows were measured in Escherichia coli B 163 and B 525 using a Coulter counter as the detector with a hydrodynamic jet focusing close to the orifice of the counter. Plotting the relative pulse height for compensated amplification of a certain size of the cells against increasing detector current, a rather sharp bend within the linear function was found, which did not occur when measuring fixed cells or polystyrene latex. The start current for transcellular ion flow causing the change of the slope is different for the potassium-deficient mutant B 525 in comparison with the wild-type B 163, indicating a change in the membrane structure of B 525 by mutation and demonstrating the sensitivity of the method for studying slight changes in membrane structure in general. The theoretical size distributions for two current values in the range of transcellular ion flow were constructed from the true size distribution at low detector currents, assuming an idealized sharp changeover of the bacterial conductivity from zero to one-third of the electrolyte conductivity

Giant culture cells by electric field-induced fusion

Because of the many potential applications in cell membrane research, genetic mapping and somatic hybridization (e.g., production of hybridoma cells), the development of techniques for in vitro cell fusion is receiving ever increasing attention [l-5]. However, cell fusion induced by the usual means (e.g., glycerol-monooleate, polyethylene glycol, Sendai virus), suffers from several disadvantages, i.e., unphysiological conditions, lack of control of the fusion process under the microscope, large variability in the number of cells subjected to fusion, low yield of fused cells, loss of intracellular components and limited viability. A new method for cell-to-cell fusion based on electrical breakdown in the membrane contact zones of two cells attached to each other has been introduced [6-14]. This method eliminates most of the disadvantages of the chemical- and virus-induced fusion techniques. Electric field-induced cell-to-cell fusion is performed in two steps. 1) Tight membrane contact between cells is achieved by dielectrophoresis [15,16], i.e., by movement of the cells under the influence of a non-uniform, alternating electric field of low intensity. This results in the formation of so-called pearl chains, the length of which depends on the cell suspension density and the inhomogeneity of the field. (2) Fusion between cells in a pearl chain is induced by an additional external field pulse of short duration and high intensity which results in the reversible electrical breakdown of the cell mem- brane [10,17,18]. Since the dielectrophoretically collected cells are aligned parallel to the field, electrical breakdown predominantly occurs in the contact zone between any two cells. After breakdown, the fusion process takes place within seconds to several minutes, depending on the species. This technique has been successfully applied in the fusion of plant protoplasts of different species, sea urchin eggs and human erythrocytes [6-14]. In the latter case giant fused cells of up to 1 mm diameter were obtained. Here, we report on electric field-induced fusion of a permanent mammalian cell line in order to test the potential of this method for somatic hybridization and for the production of giant cells from mammalian cells which could then be impaled with microelec- trodes. Friend cells (erythroblasts obtained by trans- formation with Friend virus) were used because the biochemical activity and, in turn, viability of the fused cells could easily be determined by means of the dimethylsulfoxide (DMSO)-induced haemogglobin synthesis

Dielectric Breakdown of Cell Membranes

With human and bovine red blood cells and Escherichia coli B, dielectric breakdown of cell membranes could be demonstrated using a Coulter Counter (AEG-Telefunken, Ulm, West Germany) with a hydrodynamic focusing orifice. In making measurements of the size distributions of red blood cells and bacteria versus increasing electric field strength and plotting the pulse heights versus the electric field strength, a sharp bend in the otherwise linear curve is observed due to the dielectric breakdown of the membranes. Solution of Laplace's equation for the electric field generated yields a value of about 1.6 V for the membrane potential at which dielectric breakdown occurs with modal volumes of red blood cells and bacteria. The same value is also calculated for red blood cells by applying the capacitor spring model of Crowley (1973. Biophys. J. 13:711). The corresponding electric field strength generated in the membrane at breakdown is of the order of 4 · 10(6) V/cm and, therefore, comparable with the breakdown voltages for bilayers of most oils. The critical detector voltage for breakdown depends on the volume of the cells. The volume-dependence predicted by Laplace theory with the assumption that the potential generated across the membrane is independent of volume, could be verified experimentally. Due to dielectric breakdown the red blood cells lose hemoglobin completely. This phenomenon was used to study dielectric breakdown of red blood cells in a homogeneous electric field between two flat platinum electrodes. The electric field was applied by discharging a high voltage storage capacitor via a spark gap. The calculated value of the membrane potential generated to produce dielectric breakdown in the homogeneous field is of the same order as found by means of the Coulter Counter. This indicates that mechanical rupture of the red blood cells by the hydrodynamic forces in the orifice of the Coulter Counter could also be excluded as a hemolysing mechanism. The detector voltage (or the electric field strength in the orifice) depends on the membrane composition (or the intrinsic membrane potential) as revealed by measuring the critical voltage in E. coli B harvested from the logarithmic and stationary growth phases. The critical detector voltage increased by about 30% for a given volume on reaching the stationary growth phase