Abstract
The use of embryonic stem cells (ESCs) and their progeny in high throughput drug discovery and regenerative medicine will require production at scale of well characterized cells at an appropriate level of purity. The adoption of automated bioprocessing techniques offers the possibility to overcome the lack of consistency and high failure rates seen with current manual protocols. To build the case for increased use of automation this work addresses the key question: “can an automated system match the quality of a highly skilled and experienced person working manually?” To answer this we first describe an integrated automation platform designed for the ‘hands-free’ culture and differentiation of ESCs in microwell formats. Next we outline a framework for the systematic investigation and optimization of key bioprocess variables for the rapid establishment of validatable Standard Operating Procedures (SOPs). Finally the experimental comparison between manual and automated bioprocessing is exemplified by expansion of the murine Oct-4-GiP ESC line over eight sequential passages with their subsequent directed differentiation into neural precursors. Our results show that ESCs can be effectively maintained and differentiated in a highly reproducible manner by the automated system described. Statistical analysis of the results for cell growth over single and multiple passages shows up to a 3-fold improvement in the consistency of cell growth kinetics with automated passaging. The quality of the cells produced was evaluated using a panel of biological markers including cell growth rate and viability, nutrient and metabolite profiles, changes in gene expression and immunocytochemistry. Automated processing of the ESCs had no measurable negative effect on either their pluripotency or their ability to differentiate into the three embryonic germ layers. Equally important is that over a 6-month period of culture without antibiotics in the medium, we have not had any cases of culture contamination. This study thus confirms the benefits of adopting automated bioprocess routes to produce cells for therapy and for use in basic discovery research.
Highlights
Embryonic stem cells (ESCs) are currently being evaluated for potential application in a number of diverse areas including regenerative medicine [1], drug discovery and development [2,3,4] or as routes for delivery of gene therapies [5]
We address a key question related to the use of stem cells in drug discovery and future development of the regenerative medicine industry [12]: ‘Can an automated system match the quality of a highly skilled and experienced person working manually?’ we describe an integrated automation platform (Fig. 1(a)) for the ‘hands-free’ expansion and differentiation of ESCs
This study has described an integrated automation platform designed for the ‘hands-free’ culture of ESCs over multiple passages and their subsequent directed differentiation into neuronal precursors
Summary
Embryonic stem cells (ESCs) are currently being evaluated for potential application in a number of diverse areas including regenerative medicine [1], drug discovery and development [2,3,4] or as routes for delivery of gene therapies [5]. The high level of interest is a consequence of the ability of ESCs to self renew indefinitely and to differentiate into almost every somatic cell type [6,7,8,9,10]. Effective exploitation of ESCs is, predicated upon the ability to reproducibly derive, manipulate and efficiently differentiate these cells on a suitable scale at an appropriate level of purity [11,12,13,14,15]. The often reported lack of reproducibility in ESC processing is a consequence of the large number of processing steps involved, generally carried out using largely uncontrolled and manual operations. The effect of physical forces on cells due to fluid flow during pipetting was shown to be highly significant resulting in a large degree of culture variability among trained operators. Automation of ESC handling offers the potential to reduce such variation since applied physical forces may be tightly controlled and applied consistently throughout a culture, and from culture to culture [19]
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