Study for laser-induced single cell fusion with optical tweezers manipulation system

Abstract

Cell fusion is a process by which two or multiple cells combine to form a single entity. This process is important in numerous biological events and applications, such as tissue regeneration and cell reprogramming. Artificially induced cell fusion has been used as an effective tool in developmental biology and biomedical engineering. Cell fusion has been induced using conventional methods, such as polyethylene glycol and Sendai virus-induced cell fusion. These methods are mainly based on mass fusion technique, which causes heterogeneity and reduced spatiotemporal information at a single-cell level. The demand has increased for the specific fusion of individual cells at a single-cell level while these cells are maintained viable and healthy. In this study, a robot-tweezers cell manipulation system is investigated to achieve precise laser-induced cell fusion, which provides the basis of further research on the reprogramming of somatic cells into stem cells and the generation of cancer stem cells. The three aspects involved in this study are described as follows. First, an engineering solution for the precise fusion of specific cells at a singlecell level was proposed. A cell manipulation system was developed with integrated optical tweezers and optical scissors. With optical tweezers, we used continuous wave laser to trap and transport cells. With optical scissors, we utilized pulsed ultraviolet (UV) laser to cut and perturb the cell membrane. Experiments on fibroblasts were then performed to verify the feasibility of the proposed engineering solution for laser-induced cell fusion. Using optical tweezers, we could induce two cells to form a cell pair. This pair was then fused together by pulsed UV laser irradiation. The time-lapse imaging of cells during fusion and cytoplasmic fluorescent protein transfer between cells was then observed to determine whether or not cell fusion is successful. After fusion, a binucleate cell with two nuclei approaching each other was created. Second, a study on the laser-induced cell fusion of human embryonic stem cells (hESCs) at the single-cell level was performed with the proposed engineering fusion technique. Laser-induced fusions were both achieved between two suspending cells and multiple adherent cells. The fusion efficiency of two hESCs was investigated at different laser energy levels. The success rate of hESC fusion could exceed 50% when optimized operational parameters were used. The fused products were viable with preserved pluripotency as gauged by live cell staining and active mitosis. hESCs with somatic cells were also successfully fused. Third, the laser-induced cell fusion technique was used to create cancer stem cells (CSCs). CSCs are a rare population of tumor cells with stem cell properties. These cells function as target cells used to investigate tumor growth, resistance, and recurrence in cancer biology. Evidence has shown that CSCs may be generated by occasional cell fusion. In this study, CSCs were produced by fusing hESCs with human liver hepatocellular carcinoma cells. The fused cells expressed protein markers from both stem cells and cancer cells; such cells also exhibited CSC-like properties with increased drug resistance. In summary, the proposed laser-induced cell fusion technique with an optical tweezers cell manipulation system can achieve specific cell fusion at a single-cell level with high selectivity and fusion efficiency. This method is particularly suitable for the fusion of loss-of-function mutant cells, which cannot migrate, recognize, adhere, or fuse to their targeted fusion partners. This method provides a new opportunity to study fusion during cell differentiation, maturation, reprogramming, and canceration.

Date of Award	15 Jul 2014
Original language	English
Awarding Institution	City University of Hong Kong
Supervisor	Dong SUN (Supervisor)