Abstract
As the basic unit of life, each cell is unique and is the product of its particular genome and environment. Understanding individual cell in numerous contexts is important because even cells with a genetically identical lineage may have different fates owing to local microenvironments and stochastic processes. Therefore, analysing a single cell allows the discovery of mechanisms that are unseen in an aggregated cell population. Single-cell analysis can provide further understanding of biological states and mechanisms governing diseases or responses to therapies. Remarkable capabilities exist with microfluidics and robotic technologies for single-cell analysis. These emerging technologies are transforming the biological sciences and have made a huge impact on the field of biomedical engineering.This thesis describes two projects aimed to detect and modify the behaviour and genome of a single cell. A microfluidic platform was established for the measurement of single cancer cell motility under physical signal stimulation. The mechanism behind the detected phenomenon was further explored. Engineering systems, including a microfluidic platform and robotic microinjection system, was used for single-cell genome engineering using clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated 9 (Cas9)-mediated gene editing technologies.
The first part of this work presents a novel microfluidic platform for probing single cancer cell migration property under periodic mechanical confinement. Mechanical confinement can serve as a physical sign for the modulation of intracellular signalling, thereby altering cancer cell migration mechanisms. A microfluidic chip equipped with a number of vertical constrictions was proposed for the production of periodic compression forces on cancer cells passing through narrow channels. The migration ability of these cancer cells was determined by applying chip with repeated vertical confinement to adherent MHCC-97L liver cancer cells and suspended OCI-AML leukaemia cells. Given the stimulation of the periodic mechanical confinement on-chip, the migration ability of cancer cells was promoted. Moreover, the migration speed increased as the stimulus was enhanced. The mechanical properties of the cells were measured by AFM nanoindentation and optical stretching tests. After confinement stimulation, the cancer cells possessed higher deformability and lower stiffness than the nonstimulating cells. The confinement stimulation altered the cell cytoskeleton, which governs migration speed. This phenomenon was determined by gene expression analysis.
The second part of this work proposes a single-cell gene editing approach by the robot-aided microinjection of the CRISPR/Cas9 system. A constant injection process involving the trapping, injection and retrieval of single cells was achieved by integrating a versatile microfluidic chip into a robotic microinjection system. The delivery of CRISPR/Cas9 system via a precise microinjection system can generate genome-modified cells in an efficient and safe manner. Knock-in and -out CRISPR/Cas9 systems were successfully delivered into single cells. In the knock-out application, a CRISPR system was designed to delete one exon of a target gene and further identify and validate the function of the target gene. In the knock-in application, a CRISPR system was designed to insert an eGFP donor vector to the AAVS1 site which can demonstrate the safety of this method. Plasmid cocktail (Cas9, small guide RNA and/or donor vector) was delivered and expressed in the same cell for efficient genetic modification at the single cell level. Results demonstrated the potential of CRISPR-mediated gene editing by microinjecting CRISPR/Cas9 plasmid cocktail to mammalian cells.
In summary, the proposed single-cell engineering technologies are essential to the detection of the causes and consequences of cell heterogeneity. The proposed on-chip single-cell migration analysis method enables the characterisation of the migration properties of cancer cells and is useful in the development of new therapeutic strategies for metastasis. This single-cell genome editing technology can benefit biomedical research and gene targeting therapy by providing isogenic cellular material.
| Date of Award | 6 Sept 2018 |
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| Original language | English |
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| Supervisor | Dong SUN (Supervisor) |