Incorporating size fractionation into an organoid production bioprocess

Abajo Lima, Nuria 2020. Incorporating size fractionation into an organoid production bioprocess. PhD Thesis, Cardiff University. Item availability restricted.

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Abstract

Recent studies showing that cancer organoids recapitulate the biology of primary cancers have driven tremendous excitement in their potential to revolutionize drug discovery and personalized medicine. Tumour heterogeneity at the genetic and phenotypic level drives differential responses to therapeutic agents, and this heterogeneity is preserved in organoids grown from colorectal cancers. The use of organoids as an alternative model system to 2D cultures is growing in popularity but there is a bottleneck to their widespread utilization. Organoids need to be produced on a large enough scale to adequately supply end users, from university researchers to pharmaceutical companies. In addition, the reproducibility of data from organoid experiments needs to be improved, in particular batch-to-batch variation needs to be minimized. Currently, manual processing results in organoids of varied size and while the majority are suitably functional, this means some organoids are too small to polarize, and some have necrotic cores and are effectively too big. The first aim of this thesis was to fractionate colorectal organoid subpopulations based on their size through the design of a fluidised bed in order to generate a consistent product that should improve drug screening assay reproducibility. The second aim was to study the molecular mechanisms underlying organoid phenotypic heterogeneity using biophysically-purified organoid subpopulations to assess whether organoid transcriptome is altered by their size. This work resulted in a fluidised bed design that was able to remove single cells from a heterogeneously-sized organoid population, however the current design will need to be improved if it were to be commercially applied. Fractionated colorectal cancer organoids responded to known colorectal cancer targeting compounds (LGK974, 5-FU and Trametinib) and drug response variability was reduced when a size-based fractionation step was included after cell expansion. The effects of organoid size on biology were studied by RNA-seq. Hypoxia appeared to be the main contributor to differences in gene expression between large and small organoids, which might indicate that oxygen and nutrients are reduced at the centre of large organoids. In addition, the data suggested that energy production and lipid metabolism pathways were selectively upregulated in larger organoids. Taken together, these findings showed that organoid biology is affected by their size and reinforces the importance of considering organoid size during the selection of drug testing assays and in the interpretation of the mechanism of action of candidate compounds.

Item Type:	Thesis (PhD)
Date Type:	Completion
Status:	Unpublished
Schools:	Biosciences
Subjects:	Q Science > Q Science (General)
Date of First Compliant Deposit:	18 March 2021
Last Modified:	10 Nov 2021 02:23
URI:	https://orca.cardiff.ac.uk/id/eprint/139774

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