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.

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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Lab on chip (LOC) systems often require the controlled movement of individual biological cells. Automated operation of these systems usually requires detectors to track individual cells. Electrical methods involving measurement of the conductivity or permittivity of regions between two electrodes are capable of providing this information. However, these detection systems can interfere with other dielectrophoretic LOC cell handling systems. Conversely optical systems are immune to electrical interference. Many LOC devices are fabricated with only the top surface of the device being transparent to light, precluding the use of transmitted optical detection. This is often due to the use of silicon, a favoured substrate. Here we present a low cost optical system suitable for detecting biological cells in microfluidic channels.A flow cell with a fluid microlayer approximately 105±10µm deep was fabricated having a 100±10µm thick glass window, and a reflective base layer. The reflective base was formed by thermal evaporation of gold onto a substrate. Particles within a microfluidic layer were epi-illuminated by a standard (red) laser DVD pickup unit. The flow cell permitted the laser beam to be focussed onto the gold reflector, and back through a beamsplitter onto a photodiode. This system was tested using polystyrene beads that were representative of biological cells. The position of the focal point significantly affected the base line reflected signal, but this micron scale position sensitivity could be overcome using the magnetic focussing coil of the DVD pickup. In this system, polystyrene beads down to 3µm in diameter were successfully detected.

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