Culme-Seymour, E.J.; (2010) Engineering the growth substrate for embryonic stem cell processing. Doctoral thesis , UCL (University College London).

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Abstract

Embryonic stem cells (ESCs) are pluripotent cells that represent a potentially unlimited supply of specialised cells for drug testing and cell therapy. However, a more robust, reproducible and efficient process is required for successful expansion and differentiation of these cells. One variable that exhibits a large effect on ESC culture is the growth substrate. Modifications to the currently accepted standard culture system have been made and a novel feeder layer system for human (h) ESC maintenance has been created in the research presented here. Standard hESC culture involves maintenance on supportive feeder layers of mitotically inactivated mouse embryonic fibroblasts (mEFs). The presence of these cocultured cells in pluripotent hESC cultures and during differentiation does pose technical challenges to large-scale production, thus there is a need for biphasic scalable coculture systems. Here, alginate modified with the RGD peptide sequence (commonly utilised for stimulated cell adhesion onto synthetic surfaces) has been used to immobilise mEFs into a biphasic culture system as a possible replacement to the traditional feeder layer. Analysis of proliferation, viability and ECM production of mEFs within alginate is described in this project, as well as results from both short- and long-term maintenance of hESCs on the modified layer. Apart from the choice of substrate, there are other variables within the culture system that affect ESC growth and lineage specification. Upon characterisation of the Young’s modulus (E) of an elastically tuneable glutaraldehyde cross-linked gelatin culture system, mouse (m) ESCs have been cultured on surfaces exhibiting varying degrees of stiffness. Investigations into the effect of E on the expression of pluripotency markers, regulation of spontaneous differentiation and efficiency of directed neuronal differentiation have been carried out. The results strongly suggest that adequate control of E may be critical in order to increase the yield of stem cell bioprocesses.

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