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

生物相容性可灭菌蠕虫凝胶：开发多能人类干细胞疗法的使能技术

• 批准号: EP/L024160/1
• 负责人: Steven Armes
• 金额: $ 86.18万
• 依托单位: University of Sheffield
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2014
• 资助国家: 英国
• 起止时间: 2014 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=EP%2FL024160%2F1
• 关键词: Biocompatible; Sterilisable; Worm; Gels; Enabling

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.

人类胚胎干（HES）细胞是多能细胞，可以自我更新，从而维持其多能性或根据培养条件而区分。感染的成年细胞可以产生与HES细胞相似的临床潜力的诱导多能量化（IPS）细胞。原则上，HES和IPS细胞在细胞疗法和再生医学中的应用具有巨大的潜力，因为它们具有分化为多种临床可用的细胞类型的能力。但是，定义明确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是由小鼠肉瘤细胞分泌的明胶蛋白混合物，由BD生物科学销售。这种复杂的混合物含有层粘连蛋白，肠蛋白，胶原蛋白和各种生长因子。它类似于在许多组织中发现的复杂细胞外环境，并被细胞生物学家用作模型的活性底物进行细胞培养研究。 Matrigel是4oC的液体，但在变暖至37oC时，它形成了纤维凝胶网络。 Matrigel以稀释的形式用作培养胚胎干细胞的附着底物，以在没有任何进料器细胞的情况下维持其多能，未分化的状态。尽管其成本很高，动物起源和批处理到批处理可重复性较差，但Matrigel仍被细胞生物学家广泛使用。但是，对于最终针对最终临床应用的广泛体外干细胞实验，迫切需要一种可靠地用作基线材料的替代替代合成胶合成分。此外，人们普遍认为，人类多能干细胞为细胞疗法的有效翻译将需要开发标准化测试以达到产品一致性，稳定性，毒性和免疫原性。借助这笔赠款，我们将基于生物相容性甲基丙烯酸块共聚物蠕虫样颗粒的自组装开发一系列新型的，全合成的水凝胶，这些粒子很容易在浓溶液中制备。这样的凝胶具有高度生物相容性，与许多水凝胶不同，可以简单地通过冷超滤进行灭菌：这是可能的，因为蠕虫冷却至5oC时，蠕虫会变成自由流动的球形纳米颗粒，并在返回到环境温度时转换为5oC和改革蠕虫凝胶。在为期一年的非正式合作中，我们进行了概念验证研究（参见A. Blanazs等，Jacs，2012，11，134，9741），并提交了U.Sheffield专利申请，因此我们已经具有强大的背景IP位置。但是，还有许多剩余的技术挑战，现在需要一致的跨学科研究工作来克服这些问题。我们的新蠕虫凝胶预计将取代母质（以及相关的动物衍生材料），作为人类干细胞长期储存，操纵和增殖的最方便培养基，同时保留其多能状态。 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 Moore教授将领导干细胞研究。我们要求资金支持两位经验丰富的博士后研究科学家，以在该项目上密切合作。我们已经确定了这项EPSRC赠款的两个适当的工业合作伙伴。 Geo是一家总部位于英国的特种化学公司，它将提供合成块共聚物蠕虫所需的单体构件，协助扩大研究，并在未来商业化的情况下充当原材料供应商。 Plasticell是英国的生物技术中小企业，专门从事干细胞技术。理想情况下，该公司可以帮助我们评估和优化蠕虫凝胶，以确保它们为干细胞生物学家提供适当的技术解决方案。这两家公司分别承诺提供5英镑的现金，以提供EPSRC要求的10英镑供款。

项目成果

Thermoreversible Block Copolymer Worm Gels Using Binary Mixtures of PEG Stabilizer Blocks

• DOI: 10.1021/acs.macromol.8b02491
• 发表时间: 2019-02
• 期刊: Macromolecules
• 影响因子: 5.5
• 作者: N. Penfold;Jessica R. Whatley;S. Armes

Probing the mechanism for hydrogel-based stasis induction in human pluripotent stem cells: is the chemical functionality of the hydrogel important?

• DOI: 10.1039/c9sc04734d
• 发表时间: 2019-11-11
• 期刊: Chemical science
• 影响因子: 8.4
• 作者: Sponchioni M;O'Brien CT;Borchers C;Wang E;Rivolta MN;Penfold NJW;Canton I;Armes SP
• 通讯作者: Armes SP

Disulfide-Based Diblock Copolymer Worm Gels: A Wholly-Synthetic Thermoreversible 3D Matrix for Sheet-Based Cultures.

• DOI: 10.1021/acs.biomac.5b01266
• 发表时间: 2015-11
• 期刊: Biomacromolecules
• 影响因子: 6.2
• 作者: Karen A. Simon;N. Warren;B. Mosadegh;M. R. Mohammady;G. Whitesides;S. Armes

Can percolation theory explain the gelation behavior of diblock copolymer worms?

• DOI: 10.1039/c8sc02406e
• 发表时间: 2018-09-21
• 期刊: Chemical science
• 影响因子: 8.4
• 作者: Lovett JR;Derry MJ;Yang P;Hatton FL;Warren NJ;Fowler PW;Armes SP
• 通讯作者: Armes SP

Mucin-Inspired Thermoresponsive Synthetic Hydrogels Induce Stasis in Human Pluripotent Stem Cells and Human Embryos.

• DOI: 10.1021/acscentsci.5b00370
• 发表时间: 2016-02-24
• 期刊: ACS central science
• 影响因子: 18.2
• 作者: Canton I;Warren NJ;Chahal A;Amps K;Wood A;Weightman R;Wang E;Moore H;Armes SP
• 通讯作者: Armes SP