Biocompatible Sterilisable Worm Gels: An Enabling Technology for the Development of Pluripotent Human Stem Cell-based Therapies

人类胚胎干细胞（hES）是多能细胞，可以自我更新，从而保持其多能性，或分化，这取决于培养条件。诱导多能干细胞（iPS），提供类似的临床潜力的hES细胞，可以通过感染成年细胞产生。原则上，hES和iPS细胞在细胞治疗和再生医学中的应用提供了巨大的潜力，因为它们具有分化成多种临床有用细胞类型的先天能力。然而，明确定义的培养条件对于实现hES和iPS细胞的生物医学潜力至关重要。Matrigel是一种由小鼠肉瘤细胞分泌的凝胶状蛋白混合物，由BD Biosciences销售。这种复杂的混合物含有层粘连蛋白、巢蛋白、胶原蛋白和各种生长因子;它类似于许多组织中发现的复杂的细胞外环境，并被细胞生物学家用作细胞培养研究的模型活性底物。Matrigel在4 ℃时是液体，但在加热到37 ℃时，它形成纤维状凝胶网络。基质胶以其稀释形式用作培养胚胎干细胞的附着基质，以在不存在任何饲养细胞的情况下维持其多能性、未分化状态。尽管其成本高、动物来源和批次间重现性差/可变，但基质胶仍被细胞生物学家广泛使用。然而，对于旨在最终临床应用的广泛的体外干细胞实验，迫切需要一种可可靠地用作基线材料的替代性全合成胶凝组合物。此外，人们普遍认为，将人类多能干细胞有效转化为细胞疗法将需要开发产品一致性、稳定性、毒性和免疫原性的标准化测试。借助该资助，我们将开发一系列基于生物相容性甲基丙烯酸嵌段共聚物蠕虫状颗粒自组装的新型全合成水凝胶，这些颗粒易于在浓缩水溶液中制备。这种凝胶具有高度的生物相容性，并且与许多水凝胶不同，可以简单地通过冷超滤容易地灭菌：这是可能的，因为蠕虫在冷却至5 ℃时转化为自由流动的球形纳米颗粒，并且在返回环境温度时改革蠕虫凝胶。在长达一年的非正式合作中，我们进行了概念验证研究（见A。Blanazs等人，JACS，2012，134，9741）并且提交了U.谢菲尔德专利申请，因此我们已经有了强大的背景知识产权的地位。然而，还有许多剩余的技术挑战，现在需要协调一致的跨学科研究努力来克服这些问题。我们的新蠕虫凝胶有望取代Matrigel（和相关的动物源性材料），成为人类干细胞长期储存、操作和增殖的最方便介质，同时保持其多能性状态。Steve Armes教授将领导这项跨学科研究的合成聚合物化学方面，而Harry摩尔教授将领导干细胞研究。我们要求资助两名经验丰富的博士后研究科学家在这个项目上密切合作。我们已经确定了两个合适的工业合作伙伴，为这个EPSRC赠款。GEO是一家总部位于英国的特种化学品公司，将提供合成嵌段共聚物蠕虫所需的单体结构单元，协助扩大规模研究，并在未来商业化的情况下担任原材料供应商。Plasticell是一家总部位于英国的生物技术中小企业，专门从事干细胞技术。该公司是帮助我们评估和优化我们的蠕虫凝胶的理想选择，以确保它们为干细胞生物学家提供适当的技术解决方案。这两家公司各自承诺提供5000英镑现金，以提供EPSRC所需的10000英镑捐款。

Human embryonic stem (hES) cells are pluripotent cells that can either self-renew, thereby maintaining their pluripotency, or differentiate depending on the culture conditions. Induced pluripotent stem (iPS) cells, which offer similar clinical potential to hES cells, can be generated by infecting adult cells. In principle, the application of hES and iPS cells in cell therapy and regenerative medicine offers tremendous potential because of their innate ability to differentiate into multiple, clinically-useful cell types. However, well-defined culture conditions are essential for realising the biomedical potential of hES and iPS cells. Matrigel is a gelatinous protein mixture secreted by mouse sarcoma cells and is marketed by BD Biosciences. This complex mixture contains laminin, entactin, collagen and various growth factors; it resembles the complex extracellular environment found in many tissues and is used by cell biologists as a model active substrate for cell culture studies. Matrigel is a liquid at 4oC, but on warming to 37oC it forms a fibrillar gel network. In its diluted form, Matrigel is used as an attachment substrate for culturing embryonic stem cells to maintain their pluripotent, undifferentiated state in the absence of any feeder cells. Despite its high cost, animal origin and poor/variable batch-to-batch reproducibility, Matrigel is nevertheless widely used by cell biologists. However, an alternative wholly synthetic gelling composition that can be reliably employed as a baseline material is urgently required for a wide range of in vitro stem cell experiments aimed at eventual clinical applications. Moreover, it is widely accepted that the effective translation of human pluripotent stem cells into cell therapies will require the development of standardised tests for product consistency, stability, toxicity and immunogenicity. With the aid of this grant, we will develop a range of novel, wholly synthetic hydrogels based on the self-assembly of biocompatible methacrylic block copolymer worm-like particles, which are readily prepared in concentrated aqueous solution. Such gels are highly biocompatible and, unlike many hydrogels, can be readily sterilised simply by cold ultrafiltration: this is possible because the worms transform into free-flowing spherical nanoparticles when cooled to 5oC and reform worm gels on returning to ambient temperature. In a year-long informal collaboration, we have conducted proof-of-concept studies (see A. Blanazs et al., JACS, 2012, 134, 9741) and filed a U. Sheffield patent application, thus we already have a strong background IP position. However, there are many remaining technical challenges and a concerted inter-disciplinary research effort is now required to overcome these problems. Our new worm gels are expected to replace Matrigel (and related animal-derived materials) as the most convenient medium for the long-term storage, manipulation and proliferation of human stem cells while retaining their pluripotent state. Prof. Steve Armes will lead on the synthetic polymer chemistry aspects of this inter-disciplinary study, while Prof. Harry Moore will lead on the stem cell research. We request funding to support two experienced post-doctoral research scientists to work in close collaboration on this project. We have identified two appropriate industrial partners for this EPSRC grant. GEO is a UK-based speciality chemicals company that will provide the monomer building blocks required for the synthesis of the block copolymer worms, assist with the scale-up studies and act as a raw materials supplier in the event of future commercialisation. Plasticell is a UK-based biotech SME specialising in stem cell technologies. This company is ideally placed to help us assess and optimise our worm gels to ensure that they provide an appropriate technical solution for stem cell biologists. These two companies have each pledged £ 5 K cash to provide the £ 10 K contribution required by EPSRC.