SignificanceHeart disease is the leading cause of death in the United States, yet research is limited by the inability to culture primary cardiac cells. Cardiomyocytes (CMs) derived from human induced pluripotent stem cells (iPSCs) are a promising solution for drug screening and disease modeling.AimInduced pluripotent stem cell-derived CM (iPSC-CM) differentiation and maturation studies typically use heterogeneous substrates for growth and destructive verification methods. Reproducible, tunable substrates and touch-free monitoring are needed to identify ideal conditions to produce homogenous, functional CMs.ApproachWe generated synthetic polyethylene glycol-based hydrogels for iPSC-CM differentiation and maturation. Peptide concentrations, combinations, and gel stiffness were tuned independently. Label-free optical redox imaging (ORI) was performed on a widefield microscope in a 96-well screen of gel formulations. We performed live-cell imaging throughout differentiation and early to late maturation to identify key metabolic shifts.ResultsLabel-free ORI confirmed the expected metabolic shifts toward oxidative phosphorylation throughout the differentiation and maturation processes of iPSC-CMs on synthetic hydrogels. Furthermore, ORI distinguished high and low differentiation efficiency cell batches in the cardiac progenitor stage.ConclusionsWe established a workflow for medium throughput screening of synthetic hydrogel conditions with the ability to perform repeated live-cell measurements and confirm expected metabolic shifts. These methods have implications for reproducible iPSC-CM generation in biomanufacturing.
Induced pluripotent stem cells (iPSC) can be differentiated into cardiomyocytes (CM) for disease modeling and drug screening. Batch variability in differentiated cells frequently occurs due to heterogeneity in commercial culture substrates. We performed optical metabolic imaging (OMI) on synthetic hydrogels of varying stiffnesses and compositions and observed expected metabolic shifts as iPSCs differentiated. OMI revealed metabolic differences between cells cultured on heterogenous commercial substrates and synthetic hydrogels and can be used to monitor cell sensitivity to the microenvironment. These data demonstrate OMI is a powerful tool for identifying iPSC differentiation and maturation conditions, crucial in stem cell manufacturing.
Induced pluripotent stem cells (iPSC) can generate patient-specific disease models and drug screening platforms. Heterogeneity in commercial culture substrates causes batch variation in manufactured iPSC cardiomyocyte differentiation and maturation levels. We performed optical metabolic imaging (OMI) of iPSC-cardiomyocytes on polyethylene glycol hydrogels of varying stiffnesses and adhesion ligands to assess the metabolic co-factors NAD(P)H and FAD during differentiation and maturation. Optical redox ratio at 6 days post-differentiation identified hydrogel features most favorable for subsequent cardiomyocyte maturation. These findings indicate that OMI enables rapid, non-invasive screens of favorable culture conditions during early differentiation and may translate to the biomanufacturing industry.
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