KNIGHT, ELEANOR,GRACE (2013) Influence of the micro-environment on the maintenance and differentiation of pluripotent stem cells. Doctoral thesis, Durham University.
|PDF - Accepted Version|
Routine cell culture in two dimensions forces cells to adopt an unnatural flattened morphology. This is unrepresentative of their in vivo microenvironment. When primary cells are placed onto standard tissue culture plastic, they tend to become over-proliferative, they stretch out across the rigid surface and become overly adherent to the substrate. Since the development of the glass Petri dish in the early 20th century, standard 2D cell cultureware has been optimised for a range of adherent cells to best mimic their in vivo microenvironment. This has often involved the modification of the surface to enhance cell adhesion. However a flat rigid surface that allows for limited cell interactions is not a true representation. Three dimensional (3D) cell culture models are necessary to better understand cell behaviour in vitro and to mimic the in vivo microenvironment.
A stem cells ability to form multiple cells types and their potential use in therapies to cure diseases; however the cell culture environment is limited. The application of a 3D environment to stem cell biology is of particular interest. Developmental processes involving stem cells rely on signalling between cells to determine cell fate. An increasing effort is focused on the development of new technologies to improve the standard culture environment to allow such signalling and cell interaction to occur. Using a non-degradable porous polystyrene scaffold it has been possible to optimise the 3D culture conditions for a range of cell types including pluripotent stem cells. This knowledge has permitted the long term culture of stem cells in 3D and led to the development of a 3D neuronal differentiation model.
A modified version of the scaffold was also able to mimic the stem cell niche. This allowed for self-renewal and prolonged propagation of a range of pluripotent stem cell lines and human embryonic stem cells in 3D. The prolonged culture in 3D permitted the cells to adapt to the environment. Such adaptation was demonstrated by increased levels of pluripotency markers, a change in cell shape, and increased differentiation potential of the stem cells. This 3D culture system was also assessed as a potential alternative to the teratoma assay. Using the 3D membrane to support terminal differentiation of embryoid bodies led to complex structure formation and differentiation into all three germ layers.
|Item Type:||Thesis (Doctoral)|
|Award:||Doctor of Philosophy|
|Keywords:||cell technology; cell culture; 3D models; stem cells; differentiation; tissue; teratomas|
|Faculty and Department:||Faculty of Science > Biological and Biomedical Sciences, School of|
|Copyright:||Copyright of this thesis is held by the author|
|Deposited On:||27 Jan 2014 11:50|