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.

The objective of this project is to design a cell orientation control system using a rotating electric field. In particular, the system utilizes two electrostatic phenomena known as electrophoresis and electro-rotation. The device used for creating the electric field was designed and fabricated using the MEMS fabrication technique. The cell orientation control system also includes a vision tracking system that senses the orientation of the cell and a PID controller. Overall, the system is able to control the orientation of the cell with zero steady state error. The objective of this project has been met.

The objective of this project is to design a cell orientation control system using a rotating electric field. In particular, the system utilizes two electrostatic phenomena known as electrophoresis and electro-rotation. The device used for creating the electric field was designed and fabricated using the MEMS fabrication technique. The cell orientation control system also includes a vision tracking system that senses the orientation of the cell and a PID controller. Overall, the system is able to control the orientation of the cell with zero steady state error. The objective of this project has been met.

abstract: Volumetric cell imaging using 3D optical Computed Tomography (cell CT) is advantageous for identification and characterization of cancer cells. Many diseases arise from genomic changes, some of which are manifest at the cellular level in cytostructural and protein expression (functional) features which can be resolved, captured and quantified in 3D far more sensitively and specifically than in traditional 2D microscopy. Live single cells were rotated about an axis perpendicular to the optical axis to facilitate data acquisition for functional live cell CT imaging. The goal of this thesis research was to optimize and characterize the microvortex rotation chip. Initial efforts concentrated on optimizing the microfabrication process in terms of time (6-8 hours v/s 12-16 hours), yield (100% v/s 40-60%) and ease of repeatability. This was done using a tilted exposure lithography technique, as opposed to the backside diffuser photolithography (BDPL) method used previously (Myers 2012) (Chang and Yoon 2004). The fabrication parameters for the earlier BDPL technique were also optimized so as to improve its reliability. A new, PDMS to PDMS demolding process (soft lithography) was implemented, greatly improving flexibility in terms of demolding and improving the yield to 100%, up from 20-40%. A new pump and flow sensor assembly was specified, tested, procured and set up, allowing for both pressure-control and flow-control (feedback-control) modes; all the while retaining the best features of a previous, purpose-built pump assembly. Pilot experiments were performed to obtain the flow rate regime required for cell rotation. These experiments also allowed for the determination of optimal trapezoidal neck widths (opening to the main flow channel) to be used for cell rotation characterization. The optimal optical trap forces were experimentally estimated in order to minimize the required optical power incident on the cell. Finally, the relationships between (main channel) flow rates and cell rotation rates were quantified for different trapezoidal chamber dimensions, and at predetermined constant values of laser trapping strengths, allowing for parametric characterization of the system. / Dissertation/Thesis / Demonstration of process flow in the microvortex chip / Cell rotation in a 50 microns wide (at the neck) trapezoidal chamber,at a flow rate of 95 microliters/min at approximately 0.25 rev/s / Cell rotation in a 70 microns wide (at the neck) trapezoidal chamber,at a flow rate of 7 microliters/min at approximately 0.125 rev/s / M.S. Bioengineering 2013