Cloning by nuclear transfer using mammalian somatic cells has enormous potential application. However, cloning mammalian species through somatic cell nuclear transfer has been simply inefficient in all species in which live clones have been produced, such as ‘Dolly’ the sheep, and ‘Samrupa’ the buffalo. Most of the experiments resulted failure, and the success rate ranges from 0.1% to 3%. Developmental defects have been attributed to incomplete reprogramming of the somatic nuclei by the cloning process. Researchers have tried strategies to improve the efficiency of nuclear transfer. However, significant breakthroughs are yet to happen.
The enucleation procedure consisting of extracting reprogrammable genetic material during nuclear transfer has been linked to inefficiencies due to manual error, lack of repeatability and decreased high throughput. Conventional manual enucleation process requires a series of complicated cell rotation in three-dimensional (3D) spaces using a blunt or sharp tipped pipette, and can puncture the cell during genetic material extraction. Current methods frequently damage the cell via physical or chemical contact, and thus have low throughput. Therefore, there is a need for simple, readily automated, non-contact methods for controlled cell rotation.
Precise rotation of the suspended cells is one of the many fundamental manipulations in a wide range of biotechnological applications, such as cell injection and enucleation. Noticeably scarce from the existing rotation techniques is 3D rotation of cells on one single chip. To bridge this gap, this research presents a means of controlled cell rotation for bovine oocytes around both the in-plane (yaw) and out-of-plane (pitch) axes using a simple, low cost biochip fabricated using a mixture of conventional lithography and low-cost micro-milling. It uses a phase varying dielectrophoresis (DEP)-based electrorotation (EROT) biochip platform, which has an open-top sub-millimetre square chamber enclosed by four sidewall electrodes and two bottom electrodes to induce torque to rotate the cells about two axes, thus 3D cell rotation for the first time.
Before fabrication, phase varying DEP-based rotational electric field simulations were carried out in the designed rotation chamber. For this analysis, initial rotational fields are characterised for both in-plane and out-of-plane axes using multi-physics finite element software. Electrode shape and chamber design were optimised using realistic parameters for the medium and electrode material properties. Results showed remarkable promise to rotate dielectric particles in rotational field strengths of the order of 104 V/m. From simulations, a basic biochip design was optimised.
Within the fabricated biochip, controlled rotations around the in-plane and out-of-plane axes were demonstrated, and the electric field activation frequency range and electrokinetic properties of the bovine oocytes were characterised. Rotation was measured via video image processing with data included on electronic annex. Results show controllable rotation in steps of 5 degrees around both axes with the same chip. In experiments, the maximum rotation rate reached 150°/s in yaw axis and 45-50°/s during pitch axis, while a smooth, stable and controllable rotation rate was found below 30-40°/s. Optimum rotation rates are found for inputs of 10 Vp-p at 500-800 kHz AC frequency for yaw-axis rotation, and 10-20 Vp-p and 10-100 kHz for pitch-axis rotation.
In addition, zona intact and zona free oocytes are shown to have electrical equivalence and found no noticeable difference, generalising the bio-chips capability and results. Further, experimental results were used to validate the numerical solid shell model used in design and it was found that the bovine oocytes are highly polarizable than the surrounding medium. Finally, the dielectric properties of the oocytes were fully characterised enabling further design optimization in future, if desired.
The biochip was successfully designed, optimised and experimentally validated, and successful rotation of bovine oocytes in 3D spaces was demonstrated. These results create a platform tool for biologists to utilise enucleation with high throughput efficiency and ease. In summary, this simple, transparent, low-cost, open-top, and biocompatible biochip platform, allows further function modules to be integrated and is the foundation for more powerful cell manipulation systems.
In brief key novel aspects of the research were:
• Rotation of suspended spherical oocytes in multiple axes (3D rotation) was obtained by AC induced electric fields.
• An open top biochip was successfully fabricated to enable further processing of the rotated cell in 3D spaces.
• Bovine oocyte dielectric spectra were analysed in both in-plane and out-of-plane axes for the first time.
• Bovine oocytes were determined to behave as solid spherical spheres, rather than single spherical shells.
| Identifer | oai:union.ndltd.org:canterbury.ac.nz/oai:ir.canterbury.ac.nz:10092/9170 |
| Date | January 2014 |
| Creators | Benhal, Prateek |
| Publisher | University of Canterbury. Mechanical Engineering |
| Source Sets | University of Canterbury |
| Language | English |
| Detected Language | English |
| Type | Electronic thesis or dissertation, Text |
| Rights | Copyright Prateek Benhal, http://library.canterbury.ac.nz/thesis/etheses_copyright.shtml |
| Relation | NZCU |
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