Biomedical Microdevices

, Volume 12, Issue 5, pp 777–786 | Cite as

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

  • Andrew L. Clow
  • Paul T. Gaynor
  • Björn J. Oback
Article

Abstract

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 µm wide and 250 µm 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 µm thickness. Circular holes (“micropits”) measuring 10, 20, 30, 40 or 80 µm 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.

Keywords

Nuclear transfer Cloning Electrofusion Dielectrophoresis 

Abbreviations

DEP

Dielectrophoresis

SCNT

Somatic Cell Nuclear Transfer

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Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Andrew L. Clow
    • 1
  • Paul T. Gaynor
    • 1
  • Björn J. Oback
    • 2
  1. 1.Department of Electrical and Computer EngineeringUniversity of CanterburyChristchurchNew Zealand
  2. 2.AgResearchRuakura Research CentreHamiltonNew Zealand

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