Novel in vivo biosensors for monitoring of mammalian cell cultures

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Title: Novel in vivo biosensors for monitoring of mammalian cell cultures
Authors: Goers, Lisa
Item Type: Thesis or dissertation
Abstract: Mammalian cell cultures are used for production of biopharmaceuticals, e.g. monoclonal antibodies. Only mammalian hybridoma cells contain the pathways for antibody production, but due to their multicellular origin the cells have complex nutrient requirements. Cell growth and antibody production are limited by supply of essential nutrients such as glutamine and accumulation of toxic waste products such as lactate. Many attempts have been made at tackling these challenges, e.g. by optimising growth media to keep metabolite concentrations at optimal levels. These approaches have been hampered by our ability to monitor relevant cell culture parameters such as metabolite concentration dynamics in real time. The aim of this study is to develop a solution to this problem using a synthetic biology approach. Whole-cell bacterial biosensors for important culture parameters, glutamine, leucine, alanine and lactate, were designed, built and characterised. The biosensors were designed from natural metabolite-sensing systems, specifically the Escherichia coli Ntr regulon, Lrp regulon and lldPRD operon and the Bacillus subtilis GlnK-GlnL system. Characterisation of the biosensors in defined medium using known lactate concentrations was followed by validation in mammalian cell culture media and using cell culture samples. A lactate sensor based on the lldPRD operon showed a reliable lactate-response during initial characterisation and was chosen to determine lactate concentrations in cell culture samples in parallel with lactate analysis using a bioprofiler. Generally, the lactate concentrations from the two methods showed a good match. Data points where the results differed showed that there are some sources of error in the usage of the biosensor that could be addressed in future. The results of this study also highlight the many challenges of applying synthetic biology constructs to complex industrial contexts. The biosensors presented in this study are more generally applicable in any experimental context that requires sensing of metabolites.
Content Version: Open Access
Issue Date: Sep-2014
Date Awarded: Mar-2015
Supervisor: Polizzi, Karen Marie
Freemont, Paul
Sponsor/Funder: Biotechnology and Biological Sciences Research Council (Great Britain)
Department: Life Sciences
Publisher: Imperial College London
Qualification Level: Doctoral
Qualification Name: Doctor of Philosophy (PhD)
Appears in Collections:Life Sciences PhD theses

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