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

PI：Banerjee，Ipsita 提案编号：1547618 人类多能干细胞 (hPSC) 具有在体内产生多种不同细胞类型的独特能力，因此在改变基于细胞的疗法、疾病建模和药物发现方面具有巨大潜力。 hPSC 临床转化道路上的一个重要步骤是实施可重复、同质且可扩展的细胞培养和分化技术。 hPSC 可扩展培养的主要挑战是维持高活力和增殖而不损害细胞分化为治疗相关组织类型的能力。本研究的目的是设计一个基于材料的新型平台，以实现用于生物制造的 hPSC 的可扩展培养。除了扩大规模外，所设计的系统预计还能产生尺寸均匀的同质聚集体，这将显着减少分化的变异性，并提高生物制造的保真度。目前，普遍存在的 hPSC 放大平台基于聚合悬浮培养物，有潜力生产临床相关规模的 hPSC。 该系统仍然存在重大挑战，包括初始接种群体的活力低、自发细胞聚集导致不均匀和不均匀的聚集体，以及细胞表面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的和不必要的变化。 在这项工作中，研究人员建议通过设计用于可扩展培养 hPSC 的新型仿生水凝胶胶囊来克服这些缺点。具体来说，他们建议将模拟钙粘蛋白和非钙粘蛋白细胞间相互作用的合成生物活性肽纳入三维（3D）大孔水凝胶胶囊中，用于封装和增殖 hPSC。这些肽缀合水凝胶胶囊将被设计为通过上皮钙粘蛋白（E-钙粘蛋白）综合重建细胞与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，以选择那些支持短期活力和增殖的肽。此外，大孔胶囊将从设计的肽缀合藻酸盐合成，以促进 hPSC 聚集体的均质性。胶囊设计还将防止聚集体聚结。在替代胶囊设计中增殖的 hPSC 将具有长期活力、多能性和可扩展性的特征。与目前抑制 Rho 相关卷曲螺旋蛋白激酶 (ROCK) 通路的最先进技术相比，重建细胞间接触有望显着增强单细胞活力和克隆扩增。此外，水凝胶封装将保护细胞免受生物反应器流体动力应力的影响，从而消除培养物中剪切引起的变化。拟议的工作涵盖生物材料、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，让代表性不足的少数族裔学生参与 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.