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.
Engineered tissues offer great promise as engrafted therapies and in vitro models, but these tissues require a vascular network to retain viability at large scales. Significant efforts are focused on optimizing these in vitro vascular constructs, yet current evaluation methods require fixation and immunostaining. These destructive evaluation methods alter vascular network morphology, and cannot non-invasively monitor vascular assembly over time. Here, we demonstrate that autofluorescence multiphoton microscopy (MPM) can quantitatively assess the morphology of living 3D vascular networks without fixation, labels, or dyes. Autofluorescence MPM was used to non-invasively monitor the effect of culture conditions on 3D vascular network formation. Human embryonic stem (ES) cell-derived endothelial cells and primary human pericytes cultured in polyethylene glycol (PEG) hydrogels self-assembled into 3D vascular networks. Autofluorescence MPM of the metabolic co-enzyme NAD(P)H (excitation/emission wavelengths of 750 nm/400-460 nm) was used to quantify morphological parameters at day 6 of culture. Specifically, vessel diameter, vascular density, branch point density, and integration of endothelial cells into the network were quantified. Dynamic culture conditions (flow at 1μL/sec) led to vascular networks with higher mean vessel diameter compared to static culture (p<0.05). Standard immunohistochemistry found that vascular networks were positive for markers of endothelial cells, pericytes, and tight junctions. Scanning electron micrographs confirmed vessel lumen formation with pericytes wrapped around vessels. Dye transit of FITC-dextran through the network confirmed leaky endothelial barrier function. Our results demonstrate that autofluorescence MPM can non-invasively evaluate in vitro 3D vascular networks, and could be used for quality control of engineered tissues.
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