Abstract
Somatic cell nuclear transfer (SCNT) is a widely used cell manipulation technology with broad application prospects in livestock breeding, bioreactor development, and endangered species conservation. However, low SCNT efficiency has always been the main factor hindering its promotion. Automation and batch operation are crucial to improving cloning efficiency. Although robotic SCNT systems enabling automated operation have been developed in recent years, their reliance on single-cell serial processing—with repetitive and complex procedures—continues to hinder improvements in cloning efficiency. Microfluidic technology, due to its advantages in batch operation, provides a promising avenue for improving the efficiency of SCNT cloning. Existing studies have attempted to apply microfluidic technology to nuclear transfer and electrofusion operations, which have proved the feasibility of the method in principle, but it is difficult to meet the needs of batch and precision at the same time. In addition, current oocyte electroactivation and reconstructed embryo electrofusion procedures typically employ parallel electrodes to generate a uniform electric field. While this configuration is structurally simple, the nonspecific distribution of the electric field often leads to membrane damage in non-target regions, thereby compromising cell viability and subsequent developmental potential. Therefore, achieving batch, precise, and low-damage nuclear transfer and electrofusion remains a critical challenge. In response to the above three core demands, this thesis investigates the development of a microfluidic-based system for nuclear transfer and electrofusion.First, to enable batch operation, a microfluidic-based system for batch nuclear transfer and electrofusion was designed and successfully fabricated. In Chapter 2, the functional requirements for the microchannel imposed by nuclear transfer and electrofusion are first analyzed, and an hourglass-shaped unit is designed to integrate the capabilities of cell capture, pairing, and fusion. Subsequently, the influence of the structural parameters on the electric field distribution was investigated through finite element simulation. The structural parameters were further refined to enhance cell capture efficiency while reducing potential cellular damage. Finally, in order to achieve efficient loading and collecting of cells, an open microchannel preparation scheme was proposed, and its processing method is introduced in detail.
Secondly, to improve the accuracy of cell capture and pairing in the presence of significant size differences between oocytes and somatic cells, a cell capture strategy combining dielectrophoresis (DEP) with optical tweezers (OT) was developed to enable precise capture of oocytes and somatic cells, achieving a capture success rate of 96.7%. In Chapter 3, the principles of DEP and OT were used to analyze the mechanical mechanisms underlying cell capture. Based on this analysis, DEP was employed for oocyte capture, while OT was used for the manipulation of somatic cells. Subsequently, for oocyte capture, the effects of electrical parameters and initial loading positions on capture efficiency were investigated, guiding the selection of capture voltage and the cell loading region. For somatic cell capture, the relationship between laser power and the maximum transport velocity was experimentally determined, guiding the selection of laser power and manipulation speed. Finally, the feasibility and effectiveness of both capture methods were validated through cell capture experiments.
Thirdly, to minimize membrane damage in non-target regions, the uniform electric field between two parallel electrodes was modulated using insulating hourglass-shaped units. A localized enhanced electric field distribution was established, featuring high electric field strength at the membrane contact area and low strength in surrounding regions, thereby achieving selective electroporation of cell membranes. In Chapter 4, finite element analysis was used to simulate the electric field distribution and membrane perforation density in the microchannel, based on the cell electroporation model. Simulation results revealed that pore formation was predominantly concentrated in the membrane contact area, with significantly higher density than in other membrane regions. Compared to conventional parallel-electrode electrofusion chambers, the localized enhanced electric field within the microchannel reduced the proportion of heavily porated membrane area by 47.55%. Subsequently, membrane integrity staining validated the simulation results, while cell viability staining tests under different voltages identified the safe electroporation voltage range for our microchannel. Then, calcium ion probes were used to detect calcium ion oscillation, an essential sign of oocyte activation. Results demonstrated that the localized electric field effectively triggered calcium oscillations, further validating the electroporation simulation resluts. Finally, through parthenogenetic activation experiments and subsequent embryo cleavage rates, the optimal electroactivation/electrofusion voltage was determined. Compared with the traditional electrofusion method, the applied activation/fusion voltage was reduced by 75% through locally enhancing the electric field.
Finally, we conducted nuclear transfer and electrofusion experiments based on porcine oocytes and porcine fetal fibroblasts. A microchannel-based batch SCNT–electrofusion platform was successfully established, along with a experimental procedure. The results demonstrated a pairing efficiency of 90.56% between oocytes and somatic cells, a fusion rate of 77.3%, and a cleavage rate of 70.55%.
| Date of Award | 12 Aug 2025 |
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| Original language | English |
| Awarding Institution |
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| Supervisor | Dong SUN (Supervisor), Xin ZHAO (External Supervisor) & Gang Gary FENG (Supervisor) |