Engineering Culture Conditions of Human Stem Cell-Derived Organoids

Abstract

Cell replacement therapy has demonstrated potential in the restoration of tissue function. Stem cell-derived retinal organoids potentiate accurate and reliable in vitro human models and serve as an invaluable source of functional tissue for cell therapies to treat retinal degeneration. However, clinical translation necessitates the development of organoid generation methods that are scalable, reproducible, and amenable to automation. This thesis explores approaches to better control the formation of 3D retinal organoids from 2D confluent stem cell cultures. We hypothesise that methods of biophysical and biochemical modulation will enable the enhanced self-organisation of retinal organoids in a spatially defined manner. Here, we develop micropatterning methods that improve spatial control over organoid formation, paving the way for more standardised organoid generation protocols. Concurrently, we investigate biochemical modulation strategies in 2D stem cell and retinal cultures, leading to new regimens that deepen our understanding of the cues essential for 3D retinal organoid development. By utilising three stem cell lines, including two fluorescent reporter cell lines, in parallel with control cultures, we reveal the relationship between successful modulation and the initial state and self-organisation propensity of the stem cell culture. This work establishes a foundation for more controlled organoid production and provides insights into the interplay between physical and biochemical cues in stem cell fate determination and tissue morphogenesis. Importantly, all methods developed are compliant with current Good Manufacturing Practice protocols, crucial for potential clinical translation. By addressing key challenges in organoid production, including spatial control, reproducibility, and scalability, this research contributes to realising the full potential of stem cell-derived therapies for treating degenerative diseases and restoring vision to millions worldwide.