'Future-Proof' Synthetic Surfaces for the Automated Manufacture of Human Pluripotent Stem Cells

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To realise the full biomedical potential of human pluripotent stem cells (hPSCs, which include embryonic and induced pluripotent stem cells) will require industrial scale production in completely defined conditions. However, a successful combination of chemically defined substrate, defined medium, passaging method and seeding density that permits a reproducible process to be performed by automated robotic platforms has not been identified. This hampers the use of hPSCs in academia, stem cell banking, Pharma drug toxicology & screening and regenerative medicine. Our recent finding that oxygen plasma-etched polystyrene can support pluripotency (patent filed) suggests that hPSC attachment and proliferation is governed by a three-way interaction: i) the ability of specific substrate chemistries to adsorb a cocktail of molecules from the culture medium; ii) the impact of the medium and / or passaging method on cellular gene expression, likely including adhesion molecules such as integrins; and iii) the interaction between the cell and substrate-adsorbed molecules. Coupled with our recent innovations in automation-amenable hPSC culture systems, polymer micro arrays and analysis of protein-substrate interactions, we are now able to address the hypothesis that by defining the key components mediating the three-way interaction, cost-effective substrate chemistries can be identified and coated onto standard tissue culture plastics to enable automated hPSC culture. Overcoming this major technology bottleneck will greatly facilitate the translational capacity of the only highly proliferative human stem cell type capable of forming all cell types of the body. We will begin by developing 10,000 novel polymer chemistries that will be spotted in micro array format on slides. Nottingham's unique bespoked hPSC robotic culture platform will be used to evaluate chemistries that allow attachment across 10 different hPSC lines cultured in three commonly-used media. Polymer hits will be scaled to coat 24-well plates to evaluate maintenance of pluripotency during serial passage, again using robotics as this will be the only way to enable the level of processivity required. These advances in hPSC culture technology will have immediate commercial and academic utility. The second phase of work will focus on next generation improvements to the culture system. The ability of different polymers to adsorb components from the medium will be tested by desorption ionisation (a gentle desorption method) and plasma-assisted desorption ionisation (liberates firmly-bound molecules by fragmentation). In parallel, the influence of the medium on cellular gene expression will be evaluated by Next Generation Sequencing. Collaboration with Prof. Cay Kielty (Manchester) will identify interactions between the cell and the components adsorbed to the substrate. Collectively, this information will allow the hPSC culture system to be improved by identifying synergistic combinations of polymers and by selecting media additives that adsorb to optimal substrate polymer combinations & / or enhance expression of important adhesion molecules at the cell surface. We will then demonstrate full industrial utility by showing that these culture conditions allow robust automated serial passage and production of at least 1x109 hPSCs.