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

人类多能干细胞（hPSCs）具有在体内产生多种不同细胞类型的独特能力，因此在转化基于细胞的治疗、疾病建模和药物发现方面具有巨大潜力。在人类造血干细胞临床翻译的道路上，至关重要的一步是实施可重复的、同质的、可扩展的细胞培养和分化技术。大规模培养人乳头状细胞的主要挑战是维持高活力和增殖，同时不影响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种新型的基于材料的平台，以实现用于生物制造的可扩展的人造血干细胞培养。除了扩大规模外，设计的系统有望产生均匀大小的同质聚集体，这将显著减少分化的可变性，并提高生物制造的保真度。目前，普遍存在的人造血干细胞的放大平台是基于聚集体悬浮培养，它有可能在临床相关的规模上产生人造血干细胞。该系统仍然面临着重大挑战，包括初始播种群体的低活力，自发细胞聚集导致不均匀和不均匀的聚集，以及细胞表面不受控制的动态剪切力。这些挑战会限制可伸缩性，并在差异化中引入不必要的可变性。在这项工作中，研究人员建议通过设计新型仿生水凝胶胶囊来克服这些缺点，用于可扩展培养hPSCs。具体来说，他们建议在三维（3D）大孔水凝胶胶囊中加入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，用于包封和繁殖hPSCs。这些肽偶联水凝胶胶囊将被设计用来模拟细胞微环境，通过上皮钙粘蛋白（E-cadherin）合成重建细胞-细胞接触。备选肽设计和组合将在海藻酸盐阵列平台上筛选，以选择那些支持短期生存能力和增殖的肽。此外，大孔胶囊将由设计的肽偶联海藻酸盐合成，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人造血干细胞将具有长期生存能力，多能性和可扩展性。重建细胞-细胞接触有望显著提高单细胞活力和克隆扩增，超过当前抑制Rho相关卷曲卷曲蛋白激酶（ROCK）途径的技术水平。此外，水凝胶包封将保护细胞免受生物反应器流体动力应力的影响，从而消除剪切诱导的培养变化。建议的工作包括生物材料、合成多肽、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还建议利用这一跨学科项目的医疗保健相关性，让代表性不足的少数民族学生参与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.