Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.647325
Title: Characterisation of candidate human embryonic stem cell processing methods to determine amenability to controllable manufacturing for product supply
Author: Guijarro-Leach, Juan-Jose
Awarding Body: Loughborough University
Current Institution: Loughborough University
Date of Award: 2013
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Abstract:
Human embryonic stem cells (hESCs) hold great promise for cell-based therapies and drug screening applications given their capability to self-renew and their potential to differentiate into all major lineages of somatic cells in the human body. However, for hESCs to be successfully commercialised well-defined, robust, reproducible and scalable manufacturing processes need to be developed. Moreover, unlike pharmaceuticals where the final product is distinct from the host organisms responsible for its generation, hESC-based products represent the final product and hence, careful attention needs to be taken throughout the entire bioprocess to ensure the integrity of the manufactured cell populations. This study investigated the expansion of hESCs under different culture formats conferring varying degrees of process control. Initial studies focused on the development of a systematic methodology for the in-depth characterisation of the processes involved in the manufacture of cellular products to allow for the identification, description and risk assessment of critical manufacturing variables capable of affecting processing outputs. A lack of knowledge regarding the required product specification necessary to confirm the generation of high quality cell populations was identified. To address this, several studies were performed to investigate the baseline levels at which several quality markers are expressed during the routine expansion of hESCs under different culture conditions. Feeder-free colony cultures using a defined medium were found to yield higher cell numbers per surface area available for growth. However, this culture format resulted in less reproducible processing outcomes when compared to two single cell dissociation culture formats. These results represent a data set from which product specifications can be drawn iv for the large-scale production of hESCs. To this end, and due to the limitations of growing hESCs on flat surfaces, the expansion of hESCs in a microcarrier-based culture was investigated. Preliminary microcarrier screen studies identified Hillex II as the optimal microcarrier for hESC expansion given the high cell yields obtained. Further analysis of this microcarrier identified the interactions between three culture parameters (seeding density, microcarrier mass and media volume) and allowed for the determination of the initial culture conditions to be used in agitated experiments. A novel automated microbioreactor was used for the expansion of hESCs under stirring conditions. However, the hydrodynamic environment within the microbioreactors necessary to just suspend the microcarriers was found to be detrimental for the growth of hESCs but not for the maintenance of pluripotency.
Supervisor: Not available Sponsor: EPSRC ; UKSCB
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.647325  DOI: Not available
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