High-yield electrofusion of cells using electric-field constriction

Washizu, Masao; Tsuda, Kazuhiko; Kurosawa, Osamu; Murat, Gaël

doi:10.1109/mhs.2007.4420823

Cited by 4 publications

(4 citation statements)

References 10 publications

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“…Moreover, ellipse shaped particles without a fluorescent signal are possibly cell fragments escaped from leaky electroporated cells. The established efficiency is comparable to the efficiency of both commercially available methods 56 – 58 and on-chip methods 55 , 59 – 61 . To better compete with these existing methods, there are several realistic opportunities to improve the electrofusion efficiency.…”

Section: Discussionmentioning

confidence: 73%

Electrofusion of single cells in picoliter droplets

Schoeman

Beld

Kemna

et al. 2018

Sci Rep

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We present a microfluidic chip that enables electrofusion of cells in microdroplets, with exchange of nuclear components. It is shown, to our knowledge for the first time, electrofusion of two HL60 cells, inside a microdroplet. This is the crucial intermediate step for controlled hybridoma formation where a B cell is electrofused with a myeloma cell. We use a microfluidic device consisting of a microchannel structure in PDMS bonded to a glass substrate through which droplets with two differently stained HL60 cells are transported. An array of six recessed platinum electrode pairs is used for electrofusion. When applying six voltage pulses of 2–3 V, the membrane electrical field is about 1 MV/cm for 1 ms. This results in electrofusion of these cells with a fusion yield of around 5%. The operation with individual cell pairs, the appreciable efficiency and the potential to operate in high-throughput (up to 500 cells sec−1) makes the microdroplet fusion technology a promising platform for cell electrofusion, which has the potential to compete with the conventional methods. Besides, this platform is not restricted to cell fusion but is also applicable to various other cell-based assays such as single cell analysis and differentiation assays.

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Section: Discussionmentioning

confidence: 73%

Electrofusion of single cells in picoliter droplets

Schoeman

Beld

Kemna

et al. 2018

Sci Rep

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“…After counting on average 500 droplets per experiment (n = 5), of which 15% (75 droplets) contained two cells and qualified for possible electrofusion, 19 successful electrofusion events were found resulting in 5 % electrofusion efficiency. The established efficiency is comparable to the efficiency of both commercially available methods [53][54][55] as well as on-chip methods 52,[56][57][58] . To compete with existing methods, the electrofusion efficiency must be further improved, and there are several realistic opportunities for this.…”

Section: Resultsmentioning

confidence: 79%

“…(The imaginary part of ( ) relates to the electro rotational force 58 .) Examining the expression, the real part of ( ) is bounded by limits − Adapted from ref.…”

Section: -76mentioning

confidence: 99%

“…With positive DEP the cells align towards the region of the highest electric field, see figure 3. At higher frequencies (~1-100 MHz), the field will bridge the membrane and the CM factor will compare the permittivity of the cytoplasm and the media, resulting in negative DEP at higher solution conductivities and positive DEP at low solution conductivities 50,[58][59][60] . Finally, the application of high frequencies (>1 GHz) will result in negative DEP, likely due to cytoplasmic proteins that impart a net permittivity lower than the surrounding medium 64 .…”

Section: Electrofusion Of Cells On Chip (Thesis Ewm Kemna)mentioning

confidence: 99%

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Droplet microfluidic platform for cell electrofusion

Schoeman¹

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A novel micropit device integrates automated cell positioning by dielectrophoresis and nuclear transfer by electrofusion

Clow

Gaynor

Oback

2010

Biomed Microdevices

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Nuclear transfer (NT) cloning involves manual positioning of individual donor-recipient cell couplets for electrofusion. This is time-consuming and introduces operator-dependent variation as a confounding parameter in cloning trials. In order to automate the NT procedure, we developed a micro-fluidic device that integrates automated cell positioning and electrofusion of isolated cell couplets. A simple two layer micro-fluidic device was fabricated. Thin film interdigitated titanium electrodes (300 nm thick, 250 microm wide and 250 microm apart) were deposited on a solid borosilicate glass substrate. They were coated with a film of electrically insulating photosensitive epoxy polymer (SU-8) of either 4 or 22 microm thickness. Circular holes ("micropits") measuring 10, 20, 30, 40 or 80 microm in diameter were fabricated above the electrodes. The device was immersed in hypo-osmolar fusion buffer and manually loaded with somatic donor cells and recipient oocytes. Dielectrophoresis (DEP) was used to attract cells towards the micropit and form couplets on the same side of the insulating film. Fusion pulses between 80 V and 120 V were applied to each couplet and fusion scored under a stereomicroscope. Automated couplet formation between oocytes and somatic cells was achieved using DEP. Bovine oocyte-oocyte, oocyte-follicular cells and oocyte-fibroblast couplets fused with up to 69% (n = 13), 50% (n = 30) and 78% (n = 9) efficiency, respectively. Fusion rates were comparable to parallel plate or film electrodes that are conventionally used for bovine NT. This demonstrates proof-of-principle that a micropit device is capable of both rapid cell positioning and fusion.

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High-yield electrofusion of cells using electric-field constriction

Cited by 4 publications

References 10 publications

Electrofusion of single cells in picoliter droplets

Electrofusion of single cells in picoliter droplets

Droplet microfluidic platform for cell electrofusion

A novel micropit device integrates automated cell positioning by dielectrophoresis and nuclear transfer by electrofusion

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