Electrofusion of cells: hybridoma production by electrofusion and polyethylene glycol.
Explore the source record for details and available documents.
Animals↗
Biomedical subjects
D A Stenger
Publications and source records attributed to D A Stenger.
Containment and growth of neuroblastoma cells on chemically patterned substrates.
Patterned substrates offer the promise of controlled positioning and directional guidance of growing neurites. Therefore, they could be useful for constructing small neuronal networks with defined geometry in vitro. We have fabricated chemically patterned substrates using self-assembled monolayer films with a lithographic mask technique and demonstrated the feasibility for geometrically patterning neuroblastoma cells in culture. N-octadecyltrichlorosilane (OTS) was chemically bonded to glass and fused silica substrates, rendering the surface hydrophobic and non-adhesive to cells. Using surface analysis techniques, we have confirmed that OTS films were true monolayers and can be photocleaved from the surface by deep UV irradiation. An adhesive pattern of n-(2-aminoethyl-3-aminopropyl)trimethoxysilane was formed on a selectively irradiated OTS surface via a deep UV lithographic procedure. The chemically patterned surface was then seeded with SK-N-SH human neuroblastoma cells, and cellular attachment and growth were monitored by optical microscopy. The use of 2-dimensional substrates supported the containment and growth of neuroblastoma cells within the pattern for at least 15 days in culture. These chemical patterns may also be useful in controlled arrangements of hearts cells or muscle cells on prosthetic implant devices.
Cell Adhesion↗
Measurement of acetylcholine receptor function in microcircuit-coupled myoblasts.
An electrophysiological measurement principle for long-term, noninvasive monitoring of the nicotinic acetylcholine receptor (nAChR) function is described. The measurement is based on the ability to record agonist-induced depolarizations of clonal myoblasts that have formed high impedance seals with extracellular microcircuit electrodes. The technique appears promising for several types of assays and environmental monitoring applications.
Animals↗
Deep UV photochemistry of chemisorbed monolayers: patterned coplanar molecular assemblies.
Deep ultraviolet (UV) irradiation is shown to modify organosilane self-assembled monolayer (SAM) films by a photocleavage mechanism, which renders the surface amenable to further SAM modification. Patterned UV exposure creates alternating regions of intact SAM film and hydrophilic, reactive sites. The exposed regions can undergo a second chemisorption reaction to produce an assembly of SAMs in the same molecular plane with similar substrate attachment chemistry. The UV-patterned films are used as a template for selective buildup of fluorophores, metals, and biological cells.
Axons↗
Modulation of a gated ion channel admittance in lipid bilayer membranes.
A future class of amperometric biosensors may utilize gated ion channels such as acetylcholine and glutamate receptors as chemical detection components. In this study, bilayer lipid membranes containing voltage-dependent anion channels (VDAC) were used to model an ion-channel-based biosensor which could continuously monitor AC amperometric changes resulting from induced changes in channel conductance. The in-phase and quadrature components of the induced alternating membrane current were monitored as a function of the applied DC offset voltage which was superimposed on the sinusoidal test voltage. The accuracy and sensitivity of the AC-measured VDAC response was dependent on the magnitude of the AC test voltage relative to the DC offset necessary for channel closure. The VDAC channel appears to be a suitable model protein for AC impedance-based biosensor fabrication.
Biosensing Techniques↗
Dipole interactions in electrofusion. Contributions of membrane potential and effective dipole interaction pressures.
The contributions of pulse-induced dipole-dipole interaction to the total pressure acting normal to the membranes of closely positioned pronase treated human erythrocytes during electrofusion was calculated. The total pressure was modeled as the sum of pressures arising from membrane potential and dipole-dipole attraction opposed by interbilayer repulsion. The dipole-dipole interaction was derived from the experimentally obtained cell polarizability. The threshold electric field amplitude necessary for fusion of pronase-treated human erythrocytes was experimentally obtained at various combinations of pulse duration, frequency, and the conductivity of the external medium. The theoretical values of the critical electric field amplitude compared favorably to the experimentally obtained threshold field amplitudes. Fusion by dc pulses may be primarily attributed to attainment of sufficiently high membrane potentials. However, with decreasing external conductivity and increasing sinusoidal pulse frequency (100 kHz-2.5 MHz), the induced dipole-dipole interactions provide the principal driving force for membrane failure leading to fusion.
Electric Stimulation↗
Human erythrocyte electrofusion kinetics monitored by aqueous contents mixing.
The kinetics of electrically induced fusion of human erythrocyte ghosts were monitored by the Tb/DPA and ANTS/DPX fluorescence fusion assays. Ghosts were aligned by dielectrophoresis using a 3-MHz 350-V/cm alternating field and were fused by single 15- or 50-microseconds electric field pulses of amplitude 2.5-5.0 kV/cm. Fusion was detected immediately after the pulse. The peak fluorescence change due to fusion was always obtained within 7 s of pulse application, and was highest for a 5.0 kV/cm 15-microseconds pulse. Probe leakage was measured separately and became apparent only 2-3 s after the initiation of fusion. Increasing pulse amplitudes produced higher fusion yields but produced more leakage from the fusion products. 50-microseconds pulses produced less fusion, resulting from a disruption of the dielectrophoretic alignment by fluid turbulence immediately after pulse application. Probe leakage was observed only when pulse application was preceded by dielectrophoresis, suggesting that close membrane positioning allows for additional membrane destabilization caused by the high field pulse. The fluorescence kinetics are interpreted using a simplified model depicting three major types of events: (a) fusion without observable leakage, (b) fusion followed by probe leakage, and (c) contact-related leakage from ghosts which do not undergo contents mixing.
Cell Fusion↗
Optimization of electrofusion parameters for efficient production of murine hybridomas.
Several factors which affect the production of murine hybridomas by electrofusion using SP2/0 myelomas were examined. These factors included the ionic composition, specific resistance and osmotic strength of the fusion medium, proteolytic pretreatment of the cells, and field strength of the single, rectangular, 15 or 20 microseconds electric pulse. Individual experiments were observed by phase microscopy, video-recorded, and subsequently analyzed. The efficiency of hybridoma formation was measured by the number of hybrid colonies which survived HAT medium selection. In most cases, the peak efficiency occurred when the pulse field was between 3 and 4 kV/cm. Ca++ and Mg++, in 0.1 and 0.5 mM concentrations, respectively, helped to prevent large-scale SP2/0 lysis following pulse application. Reduction of the osmolarity of the fusion medium allowed for an approximate two-fold increase in the hybrid production efficiency. However, other variations, including an increase of the specific resistance of the medium to 1.7 x 10(4) omega cm, and a reduction of the pulse width to 15 microseconds, allowed significantly higher efficiencies. Dispase pretreatment of the myelomas led to additional improvements when using a 15 microseconds pulse. The data suggest that for this system, the highest efficiencies are obtained by maximizing the attractive polarization forces between cells, while adjusting the ionic composition and pulse parameters to preserve myeloma viability.
Animals↗
Uptake of fluorescence-labeled dextrans by 10T 1/2 fibroblasts following permeation by rectangular and exponential-decay electric field pulses.
The uptake of fluorescence-labeled dextrans by adherent 10T 1/2 murine fibroblasts following electric field pulse application was used as a criterion for the efficiency of electropermeation. The cells in monolayers were permeated by immersing a coaxial electrode in culture dishes. The percentage of cells which exhibited fluorescence uptake following electric field pulse application was measured at independently varying pulse field strength and pulse length. Dextrans with molecular weights equal to or higher than 41,000 dalton require higher field strength or longer pulse time to penetrate the cells. There is no detectable advantage of using a rectangular pulse against using an exponential decay pulse of similar power. The uptake was proportional to the product of the pulse amplitude and duration over the experimental range of 40-950 microseconds and 0.1-14.5 kV/cm. Cell survival decreases at the upper end of this range. The result provides a direct comparison of electric parameters which so far have not been standardized with regard to cell electropermeation.
Animals↗
Kinetics of ultrastructural changes during electrically induced fusion of human erythrocytes.
The sequence of events during the electrically induced fusion of human erythrocytes was studied by rapid quench freeze-fracture electron microscopy. A single electric field pulse was used to induce fusion of human erythrocytes treated with pronase and closely positioned by dielectrophoresis. The electronic circuit was coupled to a rapid freezing mechanism so that ultrastructural changes of the membrane could be preserved at given time points. Pronase treatment enabled adjacent cells to approach each other within 15 nm during dielectrophoresis. The pulse caused a brief disruption of the aqueous boundaries which separated the cells. Within 100 msec following pulse application, the fracture faces exhibited discontinuous areas which were predominantly free of intramembranous particles. At 2 sec after the pulse, transient point defects attributed to intercellular contact appeared in the same membrane areas and replaced the discontinuous areas as the predominant membrane perturbation. At 10 sec after the pulse, the majority of the discontinuous areas and point defects disappeared as the intercellular distance returned to approximately 15 to 25 nm, except at sites of cytoplasmic bridge formation. Intramembranous particle clearing was observed at 60 sec following pulse application in discrete zones of membrane fusion.
Cell Fusion↗
Development and application of cell-based biosensors.
Biosensors incorporate a biological sensing element that converts a change in an immediate environment to signals conducive for processing. Biosensors have been implemented for a number of applications ranging from environmental pollutant detection to defense monitoring. Biosensors have two intriguing characteristics: (1) they have a naturally evolved selectivity to biological or biologically active analytes; and (2) biosensors have the capacity to respond to analytes in a physiologically relevant manner. In this paper, molecular biosensors, based on antibodies, enzymes, ion channels, or nucleic acids, are briefly reviewed. Moreover, cell-based biosensors are reviewed and discussed. Cell-based biosensors have been implemented using microorganisms, particularly for environmental monitoring of pollutants. Biosensors incorporating mammalian cells have a distinct advantage of responding in a manner that can offer insight into the physiological effect of an analyte. Several approaches for transduction of cellular signals are discussed: these approaches include measures of cell metabolism, impedance, intracellular potentials, and extracellular potentials. Among these approaches, networks of excitable cells cultured on microelectrode arrays are uniquely poised to provide rapid, functional classification of an analyte and ultimately constitute a potentially effective cell-based biosensor technology. Three challenges that constitute barriers to increased cell-based biosensor applications are presented: analytical methods, reproducibility, and cell sources. Possible future solutions to these challenges are discussed.
Antibodies↗