Chemically Defined and Biologically Active Microcarriers for Cell Expansion

用于细胞扩增的化学成分明确且具有生物活性的微载体

• 批准号: 1709179
• 负责人: Padma Gopalan
• 金额: $ 39万
• 依托单位: University of Wisconsin-Madison
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-07-01 至 2022-06-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1709179&HistoricalAwards=false
• 关键词: Chemically; Defined; Biologically; Active; Microcarriers

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.

第1部分：在过去的三十年中，非技术性的科学发现已经确定了可用于医学研究和治疗的细胞。但是，为了有效使用，这些细胞必须有效，可控和可重复地制造。在增强其所需功能的同时培养和扩展细胞对于生物制造至关重要，对于最终使用细胞来理解和治疗人类疾病至关重要。该建议的重点是生物制造，即两种重要的细胞类型，即已在500多个人类临床试验中使用的人类间充质干细胞（HMSC），以及形成人体血管的内皮细胞（EC）。 对HMSC和EC行为的调节（例如粘附，增殖和分化）的调节更好地了解和控制，可能会促进其有效的制造，并随着细胞疗法而获得成功。该研究计划将通过开发生物材料来模仿天然细胞外基质（ECM）的一部分，从而导致对细胞行为的理解，并更有效地制造治疗细胞。 这些生物材料以聚合物涂料的形式形式，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有效，定义明确的平台。可将3D细胞扩展的聚合物涂覆的“微载体”交付给细胞生物学家和生物工程师，然后可以将它们自定义以探测关键的生物学问题。通过这项研究在干细胞和组织工程上开发的动手展览将由PI和研究生在全国范围内传播。该研究计划将通过与研究生导师合作，启发和参与聚合物科学和工程研究。第2部分：技术摘要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，并开发聚合物涂层的微载体，这些微载体为细胞生物制造提供有效，定义明确的平台。这些关键的基本目标包括：i）了解局部和全球涂料组成对细胞行为的影响； ii）了解底物介导的生长因子（GF）隔离在GF依赖性细胞扩张中的作用； iii）探讨受体结合肽对受体激活和相关细胞行为的受体结合肽的影响。这些研究将导致设计有效的共聚物涂层和连接化学，以呈现受体受体簇的受体间距，以研究其对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，这项研究将与X射线光电学光谱（XPS）结合使用飞行中离子质谱（TOF-SIM）的时间，并评估表面组成中的局部和全球异质性。这项研究的结果将是通过共聚物化学化学化学定义的微载体合成，该化学可以对不同的细胞类型和培养基定制，并具有相关的生物启发的肽，可调节细胞粘附和GF隔离。我们设想了一类新的微载体，这些微载体使用可定制的聚合物涂层，降低媒体配方的目前棘手的成本，并实现功能性治疗细胞的有效，无XENO的扩展。在不使用复杂且昂贵的培养基的情况下，量身定制微载体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中进行变化，这是细胞和组织治疗中医疗保健的迅速增长。

项目成果

Customized hydrogel substrates for serum-free expansion of functional hMSCs.

用于功能性 hMSC 的无血清扩增的定制水凝胶基质。

• DOI: 10.1039/d0bm00540a
• 发表时间: 2020
• 期刊: RSC biomolecular sciences
• 作者: Le, Nhi T;Liu, Leona Tianran;Johnston, James;Krutty, John D;Templeton, Kayla M;Harms, Victoria;Dias, Andrew;Le, Hau;Gopalan, Padma;Murphy, William M.
• 通讯作者: Murphy, William M.

Polymer-Coated Magnetic Microspheres Conjugated with Growth Factor Receptor Binding Peptides Enable Cell Sorting

• DOI: 10.1021/acsbiomaterials.1c01199
• 发表时间: 2021-12-13
• 期刊: ACS BIOMATERIALS SCIENCE & ENGINEERING
• 影响因子: 5.8
• 作者: Krutty, John D.;Sun, Jian;Gopalan, Padma
• 通讯作者: Gopalan, Padma

Xeno-Free Bioreactor Culture of Human Mesenchymal Stromal Cells on Chemically Defined Microcarriers

化学成分确定的微载体上人间充质基质细胞的无异种生物反应器培养

• DOI: 10.1021/acsbiomaterials.0c00663
• 发表时间: 2021
• 期刊: ACS biomaterials science engineering
• 作者: John D. Krutty, Kevin Koesser