Chemically Defined and Biologically Active Microcarriers for Cell Expansion

第一部分： 在过去的三十年里，各种基础科学发现已经确定了可用于医学研究和治疗的细胞。然而，为了被有效地使用，这些电池必须被有效地、可控地和可再现地制造。培养和扩增细胞，同时增强其所需的功能是生物制造的关键，也是最终利用细胞来理解和治疗人类疾病的关键。该提案的重点是生物制造两种重要的细胞类型，即已用于500多项人类临床试验的人类间充质干细胞（hMSCs）和形成人类血管的内皮细胞（EC）。 开发更好的体外理解和控制hMSC和EC行为的调节，如粘附，增殖和分化，可以促进其有效的制造和增加作为细胞疗法的成功。该研究计划将通过开发生物材料来模拟天然细胞外基质（ECM）的部分，从而了解细胞行为，并更有效地制造治疗细胞。 这些聚合物涂层形式的生物材料将有助于回答细胞生物学中的关键基本问题，并为细胞生物制造提供一个有效的，定义明确的平台。用于3D细胞扩增的聚合物涂层“微载体”可以交付给细胞生物学家和生物工程师，然后他们可以定制它们来探索关键的生物学问题。通过这项研究开发的干细胞和组织工程动手展品将由PI和研究生在全国范围内传播。该研究项目将通过与研究生导师合作，激励本科生参与聚合物科学和工程研究。第二部分： 该研究计划将研究聚合物涂层的合成，以使用2D涂层回答细胞生物学中的关键基本问题，并开发聚合物涂层微载体，为细胞生物制造提供有效，明确的平台。这些关键的基本目标包括：i）理解局部和整体涂层组合物对细胞行为的影响; ii）理解基质介导的生长因子（GF）螯合对GF依赖性细胞扩增的作用;和iii）探索受体结合肽的受控二价呈递对受体活化和相关细胞行为的影响。这些研究将导致设计一种有效的共聚物涂层和连接化学，以呈现具有受控间距的受体-辅助受体簇，以研究它们对GF信号传导和相关细胞行为的影响。为了定量表征表面的功能，本研究将开发飞行时间-二次离子质谱（TOF-SIMS）与X射线光电子能谱（XPS），并评估表面组成的局部和全局不均匀性。这项研究的结果将是通过共聚物化学合成化学定义的微载体，该共聚物化学可定制为不同的细胞类型和培养基，具有调节细胞粘附和GF螯合的相关生物启发肽。我们设想了一类新的微载体，其使用可定制的聚合物涂层，降低了目前难以控制的培养基配方成本，并实现了功能性治疗细胞的高效、无异种扩增。定制微载体以支持特定细胞类型的粘附和受体活化而不使用复杂和昂贵的介质的能力将在生物制造中具有变革性，这是细胞和组织治疗中快速增长的医疗保健部分。

PART 1: NON-TECHNICALOver the past three decades, a variety of fundamental scientific discoveries have identified cells that can be used in medical research and therapy. However, in order to be effectively used, these cells must be manufactured efficiently, controllably, and reproducibly. Culturing and expanding cells while enhancing their desired function is essential for biomanufacturing, and critical for ultimate use of cells to understand and treat human disease. This proposal focuses on biomanufacturing two important cell types, namely human mesenchymal stem cells (hMSCs) that have been used in over 500 human clinical trials, and endothelial cells (ECs) that form human blood vessels. Developing a better in vitro understanding and control over regulation of hMSC and EC behaviors such as adhesion, proliferation, and differentiation may facilitate their efficient manufacturing and increased success as cell therapies. This research program will lead to understanding of cell behaviour, and to more effective manufacturing of therapeutic cells, by developing biomaterials to mimic parts of the native extracellular matrix (ECM). These biomaterials in the form of polymer coatings will aid in answering key fundamental questions in cell biology and provide an efficient, well-defined platform for cell biomanufacturing. The resulting polymer-coated "microcarriers" for 3D cell expansion can be delivered to cell biologists and bioengineers, who can then customize them to probe key biological questions. Hands-on exhibits developed on Stem Cells and Tissue Engineering through this research will be disseminated nationally by the PI's and the graduate students. The research program will serve to inspire and involve undergraduate students in polymer science and engineering research, by working with graduate student mentors. PART 2: TECHNICAL SUMMARYThis research program will study the synthesis of polymer coatings to answer key fundamental questions in cell biology using 2D coatings, and to develop polymer-coated microcarriers that provide an efficient, well-defined platform for cell biomanufacturing. These key fundamental goals include: i) understanding the influence of local and global coating compositions on cellular behaviors; ii) understanding the role of substrate-mediated growth factor (GF) sequestering on GF-dependent cell expansion; and iii) exploring the influence of controlled, divalent presentation of receptor-binding peptides on receptor activation and associated cell behavior. These studies will result in the design of an efficient copolymer coating and ligation chemistry to present receptor-coreceptor clusters with controlled spacing to study their influence on GF signaling and associated cell behaviors. To quantitatively characterize the functionality of the surface, this research will develop Time of Flight-Secondary Ion Mass Spectrometry (TOF-SIMS) in conjunction with X-ray photoelectron spectroscopy (XPS), and assess the local and global heterogeneities in surface composition. The outcome of this research will be the synthesis of chemically defined microcarriers via a copolymer chemistry that is customizable to different cell types and culture media with relevant bio-inspired peptides that regulate cell adhesion and GF sequestering. We envision a new class of microcarriers that use customizable polymer coatings, lower the currently intractable cost of media formulations, and achieve efficient, xeno-free expansion of functional therapeutic cells. The ability to tailor microcarriers to support adhesion and receptor activation of specific cell types without using complex and expensive media would be transformative in biomanufacturing, which is a rapidly growing segment of health care in cell and tissue therapy.