Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.631834
Title: Expansion and differentiation of human embryonic stem cells on an automated microwell platform
Author: Moens, N. C. H.
ISNI:       0000 0004 5357 864X
Awarding Body: University College London (University of London)
Current Institution: University College London (University of London)
Date of Award: 2014
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Abstract:
Human embryonic stem cells (hESCs) have the potential to provide a limitless supply of somatic cells that can find application in regenerative medicine and drug discovery. Having a scalable and cost-effective manufacturing process for the expansion and maintenance of hESCs is essential to achieving this potential. As hESC culture is currently a largely manual process, major challenges in the methods used concern variability and scalability. In this case bioprocess automation can be of benefit to reduce operator-dependent variation, therefore improving cell yield and quality, and enabling scaled-out manufacture. Use of automation will also help define a robust current Good Manufacturing Practice (cGMP) process suitable for production of cells for clinical application. To date, only certain aspects of embryonic stem cell culture have been successfully automated. This thesis describes the first use of an automated microwell platform for the completely ‘hands-free’ expansion and differentiation of hESCs. Two established hESC lines (Shef-3 and Shef-6) were taken and adapted to enzymatic passaging with TrypLE Express while maintaining their pluripotency. Culture conditions of the two cell lines were optimised using a statistical Design of Experiments (DoE) approach. This ensured that the correct feeder and hESC inoculation cell densities (ICDs) were used in microwell cultures to yield confluent, high quality hESCs. The initial automated expansion process was shown to result in a low yield of cells. The automated process was subsequently optimised primarily by making changes to the cell dissociation steps. Using the optimised automated expansion protocol, culture-adapted Shef-3 and Shef-6 hESCs were successfully expanded on the automated microwell platform over multiple passages. hESC yield was stable at ~ 2×105 cellscm-2 and was shown to be more consistent compared to the manual process. A minimum cell viability of 98.3% 0.7% was maintained throughout automated expansion. Cells were shown to be pluripotent after automated processing and maintained their ability to form EBs and differentiate into cells of all three germ layers. The automated microwell platform was also used to screen small molecules Dorsomorphin and SB431542 for their effect on neuronal differentiation, which is applicable in regenerative medicine. Overall this work has shown that automated microwell platforms provide a feasible route for the scaledout manufacture of hESCs for therapy or as high-throughput screening systems.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.631834  DOI: Not available
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