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

要实现人类多能干细胞（hPSCs，包括胚胎和诱导多能干细胞）的全部生物医学潜力，需要在完全确定的条件下进行工业规模生产。然而，化学定义的底物、定义的培养基、传代方法和播种密度的成功组合，允许自动化机器人平台执行可重复的过程，目前还没有确定。这阻碍了造血干细胞在学术界、干细胞库、药物毒理学和筛选以及再生医学中的应用。我们最近发现氧等离子体蚀刻聚苯乙烯可以支持多能性（专利申请），这表明hPSC的附着和增殖受三方面相互作用的支配：i)特定底物化学物质从培养基中吸附混合分子的能力；Ii)培养基和/或传代方法对细胞基因表达的影响，可能包括粘附分子，如整合素；和iii)细胞和底物吸附分子之间的相互作用。再加上我们最近在自动化hPSC培养系统、聚合物微阵列和蛋白质-底物相互作用分析方面的创新，我们现在能够通过定义介导三方相互作用的关键成分来解决这样一个假设，即可以识别具有成本效益的底物化学物质并将其涂覆在标准组织培养塑料上，从而实现hPSC的自动化培养。克服这一主要技术瓶颈将极大地促进唯一一种能够形成人体所有细胞类型的高增殖人类干细胞的转化能力。我们将从开发10000种新型聚合物化学物质开始，这些化学物质将以微阵列的形式出现在幻灯片上。诺丁汉大学独特的定制hPSC机器人培养平台将用于评估在三种常用培养基中培养的10种不同的hPSC系上的化学物质。聚合物将被涂覆在24孔板上，以评估在连续传代过程中多能性的维持情况，再次使用机器人技术，因为这将是实现所需处理水平的唯一方法。hPSC培养技术的这些进步将具有直接的商业和学术效用。第二阶段的工作将集中在下一代文化系统的改进上。不同聚合物从介质中吸附组分的能力将通过解吸电离（一种温和的解吸方法）和等离子体辅助解吸电离（通过破碎释放紧密结合的分子）进行测试。同时，培养基对细胞基因表达的影响将通过Next Generation Sequencing进行评估。与Cay Kielty教授（曼彻斯特）合作，将确定细胞与吸附在底物上的成分之间的相互作用。总的来说，这些信息将允许hPSC培养系统通过识别聚合物的协同组合和通过选择吸附到最佳底物聚合物组合和/或增强细胞表面重要粘附分子表达的培养基添加剂来改进。然后，我们将通过展示这些培养条件允许至少1x109个hPSCs的强大自动化连续传代和生产来展示完整的工业实用性。

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.