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

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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.