APPLICATION OF THREE-DIMENSIONAL IMAGING TECHNIQUES TO THE STUDY OF MULTI-DIMENSIONAL HUMAN CELL CULTURES

Abstract

Three dimensional (3D) cultures are increasingly used in preference to 2D cultures for biomedical research, as they better resemble tissues in vivo. However, due to their size and density and the time take to generate them, microscopic analysis is usually limited to a single end-point, and 3D imaging of multi-dimensional human cell cultures has only been briefly explored. This thesis examines the potential of observing 3D human cell cultures using live imaging techniques. 3D neural cultures (from induced neural stem cells (iNS)) and immortalised keratinocytes were used to establish complex cultures, and three live imaging techniques (confocal microscopy, multiphoton microscopy and light-sheet fluorescence microscopy) were compared. A unique behavioural signature was seen in the neural cells, with increased environmental rigidity promoting their propagation better than suspension cultures; morphological changes of keratinocytes and their nuclei were tracked in 3D during differentiation; and a method is proposed for 3D co-culture of neuronal and skin cultures. The technical pipeline proposed can thus be applied to different cell types and cultures, to answer biological questions in real time complex tissue-mimetic culture systems.