EAGER: Biomanufacturing: Engineered hydrogel capsules for controlled scalable cultures of pluripotent stem cells

PI：Banerjee，Ipsita 提案编号：1547618人多能干细胞（hPSC）具有在体内产生许多不同细胞类型的独特能力，因此在转化基于细胞的疗法、疾病建模和药物发现方面具有巨大潜力。hPSC临床转化途径中的重要一步是实施可重复、同质和可扩展的细胞培养和分化技术。 hPSC的可扩展培养物的主要挑战是维持高活力和增殖，而不损害细胞分化成治疗相关组织类型的能力。本研究的目的是设计一种新的基于材料的平台，以实现这种可扩展的生物制造hPSC培养。除了按比例放大外，预计所设计的系统将产生均匀尺寸的均匀聚集体，这将显著降低分化的可变性，并导致生物制造的保真度增加。目前，普遍存在的hPSC规模扩大平台是基于聚集体悬浮培养物，其具有以临床相关规模生产hPSC的潜力。 该系统仍然存在实质性挑战，包括初始接种群体的低活力，导致不均匀和不均匀聚集体的自发细胞聚集，以及细胞表面上不受控制的动态剪切力。 这些挑战可能会限制可扩展性，并在差异化方面引入不必要和不必要的可变性。 在这项工作中，研究人员提出通过设计新型仿生水凝胶胶囊来克服这些缺点，用于可扩展的hPSC培养。具体地，他们提出将模拟钙粘蛋白和非钙粘蛋白细胞-细胞相互作用的合成生物活性肽掺入三维（3D）大孔水凝胶胶囊中，用于包封和增殖hPSC。这些肽缀合的水凝胶胶囊将被设计为通过上皮钙粘蛋白（E-钙粘蛋白）合成重建细胞-细胞接触来模拟细胞微环境。将在藻酸盐阵列平台中筛选替代肽设计和组合，以选择支持短期活力和增殖的那些。此外，将从设计的肽缀合的藻酸盐合成大孔胶囊以促进hPSC聚集体的均匀性。胶囊设计还将防止聚集体的聚结。在替代胶囊设计中繁殖的hPSC将表征长期活力、多能性和可扩展性。与抑制Rho相关卷曲螺旋蛋白激酶（ROCK）途径的现有技术相比，预期重建细胞-细胞接触显著增强单细胞活力和克隆扩增。此外，水凝胶包封将保护细胞免受生物反应器流体动力学应力的影响，从而消除培养物中剪切诱导的变化。拟议的工作包括生物材料，合成肽，干细胞和生物加工，从而为来自不同学科的学生提供跨学科培训的机会。PI还建议利用这个跨学科项目的医疗保健相关性，让代表性不足和少数民族学生参与STEM领域。

PI: Banerjee, Ipsita Proposal Number: 1547618Human pluripotent stem cells (hPSCs) possess the unique capability of giving rise to many different cell types in the body and hence hold great potential in transforming cell-based therapies, disease modeling, and drug discovery. A vital step in the path to clinical translation of hPSCs is to implement reproducible, homogenous, and scalable cell culture and differentiation technologies. The primary challenge in scalable cultures of hPSCs is the maintenance of high viability and proliferation without compromising the ability of the cells to differentiate into therapeutically relevant tissue types. The objective of this research is to design a novel materials-based platform to achieve such scalable culture of hPSCs for biomanufacturing. Besides scale-up, the designed system is expected to produce homogenous aggregates of uniform size, which will significantly reduce variability in differentiation and lead to increased fidelity in biomanufacturing. Currently, the ubiquitous scale-up platform of hPSCs is based on aggregate suspension cultures which have the potential to produce hPSCs at clinically relevant scales. Substantial challenges still remain with this system, including low viability of initial seeding population, spontaneous cell aggregation leading to inhomogeneous and non-uniform aggregates, and uncontrolled and dynamic shear force on the cell surface. These challenges can restrict scalability and introduce unwanted and unnecessary variability on differentiation. In this work, the investigators propose to overcome these shortcomings through the design of novel biomimetic hydrogel capsules for scalable culture of hPSCs. Specifically, they propose to incorporate synthetic bioactive peptides mimicking cadherin and non-cadherin cell-cell interactions within three dimensional (3D) macroporous hydrogel capsules, for encapsulating and propagating hPSCs. These peptide-conjugated hydrogel capsules will be designed to mimic the cellular microenvironment by synthetically recreating cell-cell contacts through epithelial-cadherin (E-cadherin). Alternate peptide designs and combinations will be screened in an alginate array platform to select for those supporting short-term viability and proliferation. Further, macroporous capsules will be synthesized from the designed peptide-conjugated alginate to facilitate homogeneity in hPSC aggregates. The capsule design will also prevent coalescence of the aggregates. hPSCs propagated in alternate capsule designs will be characterized for long-term viability, pluripotency and scalability. Recreating cell-cell contact is expected to significantly enhance single cell viability and clonal expansion over current state-of-art of inhibiting Rho associated coiled coil protein kinase (ROCK) pathway. Furthermore, hydrogel encapsulation will protect the cells from bioreactor hydrodynamic stresses, hence removing shear-induced variations in the culture. The proposed work encompasses biomaterials, synthetic peptides, stem cells and bioprocessing, hence providing opportunities for interdisciplinary training of students from different disciplines. The PI also proposes to utilize health care relevance of this cross-disciplinary project to involve under-represented and minority students into STEM fields.