Analysis of the effects of an orifice plate on the membrane potential in electroporation and electrofusion of cells

Techaumnat, Boonchai; Washizu, Masao

doi:10.1088/0022-3727/40/6/036

Cited by 26 publications

(21 citation statements)

References 10 publications

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“…In conclusion, thanks to the development of a microstructured biochip, it has been possible to study with good accuracy the electrofusion process between two cells. The fusion observations demonstrate that the use of the positive DEP and dielectric structures allows for a maximal membrane permeabilization at the point of contact of the two cells (correlated with a recent theoretical study ). This observation is very important because it shows that in a microstructured biochip, the mixing of the two cells cytosols can be achieved with very small losses of the cell content.…”

Section: Discussionsupporting

confidence: 81%

“…This observation reinforces the finding that the permeabilization of the membrane is very intense in the contact area between the two cells and very weak in the other parts of the cell membrane. This point is interesting because it links these experiments with the theoretical calculations of Techaumnat and Washizu , showing that two cells put in contact in similar dielectric structures will have the highest membrane voltage at their contact points and a very low one on the other parts of the cell surface. We thus experimentally validate the asymmetric electropermeabilization in the dielectric microstructured biochips, a situation very different from the classical symmetric permeabilization when the dielectric microstructures are absent (see Supporting Information Fig.…”

Section: Discussionmentioning

confidence: 75%

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High‐resolution analyses of cell fusion dynamics in a biochip

2012

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In this study, we analyzed the electrofusion of two cells in a biochip that has been developed to perform the capture by dielectrophoresis and the electrofusion of pairs of cells. The good transparency of the microsystem allowed analyzing the details of the fusion events. By staining one of the cells, the mixing of the two cytosols could be observed during the electrofusion experiment. We show for the first time the rapidity of the mixing of the two cytosols: less than 5 s under our experimental conditions. By comparing these experimental results to a numerical simulation, we found that the rate of this phenomenon is compatible with a diffusion-only mechanism, showing that during the fusion, the two cell membranes in contact are affected by very rapid structural changes and do not limit the exchange of the cytosols between the two cells. A point of interest is the use of dielectric structures to concentrate the electric field and of positive dielectrophoresis to capture cells in the area where the electric field is more intense. This technique allows the increase of the cell-to-cell contact and limits cell cytosol leakages during the fusion process.

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Section: Discussionsupporting

confidence: 81%

Section: Discussionmentioning

confidence: 75%

High‐resolution analyses of cell fusion dynamics in a biochip

2012

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“…In our method, micro-slits induce electric field constriction and, in effect, membrane breakdown leading to fusion can be achieved at a low voltage with minimal cell damage 15,17 . Membrane breakdown is limited to the point of contact between the cell pairs at the micro-slits, 17 thus damage to the entire cell is minimal. In contrast, PEG method used in previous studies is known to cause considerable cell damage, which may interfere with cell cycle progression, and hence a delay in cell division 6,8 …”

Section: Discussionmentioning

confidence: 99%

Adhesion patterning by a novel air-lock technique enables localization and in-situ real-time imaging of reprogramming events in one-to-one electrofused hybrids

et al. 2016

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Although fusion of somatic cells with embryonic stem (ES) cells has been shown to induce reprogramming, single-cell level details of the transitory phenotypic changes that occur during fusion-based reprogramming are still lacking. Our group previously reported on the technique of one-to-one electrofusion via micro-slits in a microfluidic platform. In this study, we focused on developing a novel air-lock patterning technique for creating localized adhesion zones around the micro-slits for cell localization and real-time imaging of post fusion events with a single-cell resolution. Mouse embryonic fibroblasts (MEF) were fused individually with mouse ES cells using a polydimethylsiloxane (PDMS) fusion chip consisting of two feeder channels with a separating wall containing an array of micro-slits (slit width ∼3 μm) at a regular spacing. ES cells and MEFs were introduced separately into the channels, juxtaposed on the micro-slits by dielectrophoresis and fused one-to-one by a pulse voltage. To localize fused cells for on-chip culture and time-lapse microscopy, we implemented a two-step approach of air-lock bovine serum albumin patterning and Matrigel coating to create localized adhesion areas around the micro-slits. As a result of time-lapse imaging, we could determine that cell division occurs within 24 h after fusion, much earlier than the 2–3 days reported by earlier studies. Remarkably, Oct4-GFP (Green Fluorescent Protein) was confirmed after 25 h of fusion and thereafter stably expressed by daughter cells of fused cells. Thus, integrated into our high-yield electrofusion platform, the technique of air-lock assisted adhesion patterning enables a single-cell level tracking of fused cells to highlight cell-level dynamics during fusion-based reprogramming.

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“…that of water). The simulations assume that the rate of change of the electric (Techaumnat and Washizu 2007) and thus r E…”

Section: Electric Field Modelmentioning

confidence: 99%

Coplanar film electrodes facilitate bovine nuclear transfer cloning

Clow

Gaynor

Oback

2009

Biomed Microdevices

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Automated lab on chip systems offer increased throughput and reproducibility, but the implementation of microelectrodes presently relies on miniaturization of parallel plate electrodes that are time consuming and costly to fabricate. Electric field modelling of open electrofusion chambers suggested that widely spaced (> or =2 mm) coplanar film electrodes should result in similar cell fusion rates as parallel plate electrodes provided the cell positioning was roughly midway between the electrodes. This hypothesis was investigated by electrofusion trials of bovine oocyte-donor cell couplets used in nuclear transfer (NT) cloning. Comparative experiments with reference parallel plate electrodes were conducted as controls. Coplanar fusion rates > or = 90% were demonstrated for embryonic blastomeres, follicular cells and fetal and adult fibroblasts as NT donor cells. For embryonic and adult cell types, there was no significant difference in fusion rate between coplanar and parallel plate electrodes. For both electrode geometries, fusion efficiency with adult fibroblasts was highest at a calculated field strength of 2.33 kV/cm. The coplanar electrodes required a voltage pi/2 times greater than parallel plate electrodes to achieve equivalent field strength when the couplets are placed midway between the electrodes.

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Analysis of the effects of an orifice plate on the membrane potential in electroporation and electrofusion of cells

Cited by 26 publications

References 10 publications

High‐resolution analyses of cell fusion dynamics in a biochip

High‐resolution analyses of cell fusion dynamics in a biochip

Adhesion patterning by a novel air-lock technique enables localization and in-situ real-time imaging of reprogramming events in one-to-one electrofused hybrids

Coplanar film electrodes facilitate bovine nuclear transfer cloning

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