Key Characterization and Synthesis Tools for Defined Polymeric Hydrogels to Guide Cell Fate

主要目的是研究细胞外基质对细胞命运的作用，特别是设计一系列聚合物水凝胶，并研究其对细胞分化，增殖和指导的影响。 由于缺乏生物学线索，目前培养细胞的方法提供的信息有限。 这导致药物筛选中的显著假阳性（并且可能同样多的假阴性）。为了克服这一缺陷，我们建议合成仿生水凝胶用于三维细胞培养和随后的药物筛选。此外，许多细胞不能通过传统的二维技术培养，需要异种移植小鼠模型，这是昂贵和费力的。 我们的3D水凝胶策略有望克服这些缺陷，并将能够培养多种细胞类型。 我们的目标是用定义的仿生ECM克服的另一个关键挑战是移植后的细胞存活。 传统的方法使用生理盐水进行细胞递送，结果几乎所有（如果不是全部）细胞死亡。 在具有适当因子的限定水凝胶中递送细胞将导致存活率提高。 建议的设备是我们成功的关键。肽通过特定的整合素相互作用形成细胞的关键附着位点，需要肽合成仪，我们可以合成定制的功能肽。拟议的肽合成器将使用90%的溶剂，并需要几分钟而不是几个小时的耦合化学，使该系统更有效，更环保。 凝胶计数是一种关键的工具，可以用来询问我们在3D中培养的细胞。 在多伦多只有一个GelCount（在儿童医院），考虑到我们设施之间的距离以及这种距离对我们培养的细胞的影响，这对于日常使用是不实际的。 力学测试仪的升级对于我们优化力学测试仪的使用至关重要，该测试仪专门用于测量弱材料（如我们的水凝胶）的力学性能。这种相对较小的投资将为我们的知识进步提供巨大的效用。培养箱是任何细胞培养设施成功运作的关键。 我们的联合实验室有120多名高素质的人员，他们将从拟议的设备中受益。 它将为他们提供设计和评估用于生物医学工程应用的新型生物材料所需的工具。

The primary objective is to examine the role of the extracellular matrix on cell fate and specifically to design a series of polymeric hydrogels and investigate their impact on cell differentiation, proliferation and guidance. Current methods of growing cells in culture provide limited information due to lack of biological cues. This leads to significant false positives (and likely just as many false negatives) in drug screening. To overcome this deficit we propose to synthesize biomimetic hydrogels for 3-dimensional cell culture and subsequent drug screening. Moreover, many cells cannot be cultured by traditional 2-dimensional techniques, requiring xenograft mouse models, which are expensive and laborious. Our 3D hydrogel strategy promises to overcome these deficits and will enable the culture of numerous cell types. Another key challenge that we aim to overcome with a defined biomimetic ECM is cell survival after transplantation. Traditional methods use saline for cell delivery with the consequence that nearly all (if not all) of the cells die. Delivering the cells in a defined hydrogel with appropriate factors will result in improved survival. The proposed equipment is key to our success. Peptides form key attachment sites for the cells through specific integrin interactions, necessitating a peptide synthesizer with which we can synthesize customized, functional peptides. The proposed peptide synthesizer will use 90% less solvent and take minutes instead of hours for coupling chemistry, making this system more efficient and environmentally friendly. The gel count is a critical instrument with which to interrogate our cells cultured in 3D. There is only one other GelCount in Toronto (at the Hospital for Sick Children), which is unpractical for daily use given the distance between our facilities and the consequent effect this distance would have on our cultured cells. The upgrade to the mechanical tester is critical to our optimized use of the mechanical tester, which is specifically designed to measure the mechanical properties of weak materials, such as our hydrogels. This relatively small investment will provide great utility to our advancement of knowledge. The incubator is key to the successful functioning of any cell culture facility. There are over 120 highly qualified personnel in our combined laboratories who will benefit from the proposed equipment. It will provide them with the tools required to design and evaluate novel biomaterials for use in biomedical engineering applications.