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影响，特别是设计一系列聚合物水凝胶，并研究它们对细胞分化、增殖和导向的影响。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。这导致在药物筛选中出现显著的假阳性(也可能有同样多的假阴性)。为了克服这一缺陷，我们建议合成用于三维细胞培养和随后的药物筛选的仿生水凝胶。此外，许多细胞不能用传统的二维技术培养，需要昂贵而费力的异种小鼠模型。我们的3D水凝胶战略有望克服这些缺陷，并将使多种细胞类型的培养成为可能。我们致力于用一种明确的仿生细胞外基质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输送，结果是几乎所有(如果不是全部)的细胞死亡。将细胞置于特定的水凝胶中，并添加适当的因子，将会提高存活率。提议的设备是我们成功的关键。多肽通过特定的整合素相互作用形成细胞的关键附着部位，这就需要一个多肽合成器，我们可以用它来合成定制的功能性多肽。建议的多肽合成器使用的溶剂将减少90%，偶联化学需要几分钟而不是几个小时，使该系统更加高效和环保。凝胶计数是用来审问我们3D培养细胞的关键工具。多伦多只有一家GelCount(患病儿童医院)，考虑到我们设施之间的距离及其对培养细胞的影响，这在日常使用中是不切实际的。机械试验机的升级对我们优化使用机械试验机至关重要，机械试验机是专门为测量弱材料(如我们的水凝胶)的机械性能而设计的。这一相对较小的投资将为我们的知识进步提供巨大的效用。孵化器是任何细胞培养设施成功运作的关键。我们联合实验室中有120多名高素质人员，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.