Embryonic stem cells (ESCs) are promising as therapeutic material since they are pluripotent
(potentially differentiate into any mature cell) and have “limitless” self-renewal capacity. To achieve
widespread clinical utility, ESC cultures have to be designed to meet specific process requirements
(e.g. quantity, quality etc.). Currently, most pluripotent stem cell (PSC) cultures are fragmented
protocols relying on operator–intensive processing, as 2D monolayers on tissue culture plastic, at
ambient O2 conditions. Incidentally, such culture conditions are sub-optimal, often leading to
unscheduled stem cell behaviour. This thesis examines how ESC bioprocesses can be improved.
Culture environment effects on ESCs are investigated, as well as computational tools for in silico
design. I demonstrate how critical culture parameters and mathematical modelling can be exploited
to improve the undifferentiated expansion of ESCs.
Beginning with 3D murine ESCs (mESCs) cultures, 1) dynamic rotary cultures were demonstrated to
improve self-renewal signalling activity, yielding improved proliferation of mESCs with higher
“stemness” levels. 2) Culture metabolism was another critical factor. During batch feeding,
metabolites accumulate within the culture environment especially at later stages in culture, causing
stresses that impair ESC proliferation and “stemness”, independent of growth factor levels. In
contrast, perfusion feeding maintained well-regulated culture environments that promoted the
expansion of highly “naïve” mESCs. 3) Computational approaches can complement bioprocess
design. Mathematical models identified novel multi-scale interactions within the bioprocess and
effectively simulated bioreactor fluid dynamics. 4) As a means to further optimize the bioprocess,
alternative signalling factors were combined with dynamic perfusion cultures in reduced (5%) O2
conditions, which generated increased cell yields having high “stemness” levels at half the costs.
In conclusion, numerous ‘standard’ culture conditions were found to be sub-optimal for mESC
culture, emphasizing the need for improved bioprocesses using rational design based on stem cell
bioscience. It is anticipated that these integrated stem cell bioprocesses, can improve product yield
and quality at reduced costs. Such bioprocess strategies will facilitate the usage of PSCs as
therapeutics.