Chemically Defined and Biologically Active Microcarriers for Cell Expansion

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，为了有效地使用，这些细胞必须高效、可控和可复制地制造。培养和扩增细胞，同时增强其所需功能，对于生物制造至关重要，对于最终使用细胞来了解和治疗人类疾病至关重要。该提案侧重于生物制造两种重要的细胞类型，即已在500多项人体临床试验中使用的人类间充质干细胞（hMSCs）和形成人类血管的内皮细胞（ECs）。在体外更好地了解和控制hMSC和EC的粘附、增殖和分化等行为的调节，可能有助于它们的有效制造和作为细胞疗法的成功率提高。该研究项目将通过开发生物材料来模拟部分天然细胞外基质（ECM），从而导致对细胞行为的理解，并更有效地制造治疗细胞。这些高分子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高效、明确的平台。由此产生的用于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关于干细胞和组织工程的实践展览将由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，激励和参与高分子科学与工程研究的本科生。本研究项目将研究聚合物涂层的合成，以回答使用2D涂层的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高效，明确的平台。这些关键的基本目标包括：1)了解局部和全局涂层成分对细胞行为的影响；ii)了解底物介导的生长因子（GF）隔离对GF依赖性细胞扩增的作用；iii)探索受控制的受体结合肽的二价呈现对受体激活和相关细胞行为的影响。这些研究将导致设计一种高效的共聚物涂层和连接化学来呈现具有控制间距的受体-辅受体簇，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，本研究将结合x射线光电子能谱（XPS）开发飞行时间二次离子质谱（TOF-SIMS），并评估表面成分的局部和全局异质性。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，该共聚物化学可针对不同的细胞类型和培养基定制，并具有调节细胞粘附和GF隔离的相关生物启发肽。我们设想了一种新型的微载体，它使用可定制的聚合物涂层，降低目前难以解决的介质配方成本，并实现功能性治疗细胞的高效、无xeno扩展。在不使用复杂和昂贵的介质的情况下，定制微载体以支持特定细胞类型的粘附和受体激活的能力将在生物制造领域发生变革，生物制造是细胞和组织治疗中快速增长的医疗保健领域。

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.