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

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.

The technology innovations required to develop fully defined, automation-amenable hPSC culture systems will have far reaching impact on industrial utility, drug screening & toxicology and regenerative medicine, as well as student / staff training opportunities & public perception of science. Industrial Impact We will ensure industrial engagement and technology exploitation by working with the University of Nottingham's Research Innovation Service (RIS). This department has a wealth of experience in identifying IP opportunities, licensing, establishing spin-out companies from researchers' technologies and negotiating deals with Pharma. For example, RIS identified IP opportunities from the synthetic substrates Denning / Alexander developed for pluripotent stem cell culture. RIS organised distribution of non-confidential flyers to major plasticware companies, as well as the subsequent provision of samples to BD Biosciences (under Material Transfer Agreement) and the teleconference with Thermo (under a Confidentiality Agreement; see letter). University-wide, RIS has been responsible for filing numerous patents (63 patents filed Sept '06 - Mar '08) and spinning out in excess of 30 companies, which generate an annual income averaging over 700,000. Davies was involved in establishing three spin-outs, Molecular Profiles, Critical Pharmaceuticals and Regentec. The applicants already have strong links with industry. Applicants are members of the Bioprocess Industry Club (BRIC) that is underpinned by 17 companies including GSK, Lonza and Novozymes. Funding is held with industry for safety screening, including Syngenta (via a BBSRC industrial partnership award) and GSK, AZ and Roche (via a Stem Cells for Safer Medicine award). Impact will also be facilitated via collaboration. Polymer array technology will be developed with Biodot and Scienion (see letter), the suppliers of instrumentation, while automation of hPSC culture will be developed with Tecan Robotics. Impact to the academic community will be readily achieved through our membership of the International Stem Cell Initiative and our relationship with the UK Stem Cell Bank; we have already deposited two human embryonic stem cell lines to this facility. Training Our repertoire of subject-specific training includes taught Masters Courses in Stem Cell Technology, Nanoscience and in Advanced and Genomic and Proteomic Sciences, an EPSRC-funded Doctoral Training Centre and a track record of supplying skilled workers to the industrial sector. Past students and postdocs now work for a range of institutions, such as Pharma (e.g. GSK, AZ, Syngenta, Pfizer), Stem Cell Banks (e.g. Anthony Nolan trust for cord blood banking, Indian Cord Blood Bank, UK Stem Cell Bank) and medicine. The work outlined in this proposal will not only provide new technology / knowledge with which to teach postgraduate studies, but also provide highly trained PDRAs in this area. As examples, four members of Alexander's group have gone on to take up Lectureships and many more have moved into industry. Of the 60 PhD students & 30 postdoctoral fellows mentored by Davies, 5 have senior positions within the pharmaceutical industry, 5 are Advanced Research Fellows, 14 have been appointed to academic positions within leading international Universities & 4 are full Professors. Public engagement of science Applicants have a track record in high impact manuscripts & presenting at international conferences. Engagement of the public is through television (BBC 6 o'clock news, Horizon), radio (Material World), newspapers (Telegraph, Daily Mail), magazines (Preclinical World, BBSRC Business, International Hospital Equipment & Solutions) and the University Research Television website [www.research-tv.com]. Outreach activities include hosting 'A' level students or taking science to them. These activities are facilitated by the University of Nottingham Press & Media office or the London Science Media Centre.