Label-Free\nRaman Spectroscopy for Assessing Purity\nand Maturation of hiPSC-Derived Cardiac Tissue

I. Background: Human\ninduced pluripotent stem cell (hiPSC) derived\ncardiomyocytes (CMs) have been utilized in drug toxicity evaluation,\ndrug discovery, and treating heart failure patients, showing substantial\neffects. Ensuring the quality, purity, and maturation of hiPSC-CMs\nduring large-scale production is crucial. There is a growing demand\nfor a novel method to characterize cell molecular profiles without\nlabels and without causing damage. II. Methods: In this study, we\nemployed label-free Raman microscopy to evaluate hiPSC-derived CMs.\nThe study involved the characterization of cell molecular profiles\nwithout labels and without causing damage. The correlation between\nRaman spectroscopy of specific components, such as cytochrome <i>c</i> and myoglobin, and CM purity and maturation following\nhiPSC differentiation was investigated. Additionally, the validation\nof this correlation was performed by assessing mixtures of commercially\navailable CMs (iCell cardiomyocytes2) and fibroblasts at various ratios\nas well as hiPSC-derived CMs with different efficiencies. Furthermore,\nCMs were matured using rapid pacing of traveling waves, and the Raman\nprofiles of matured CMs were compared to those of immature ones. III.\nResults: Raman spectroscopy indicated that the cytochrome <i>c</i> and myoglobin showed correlation with the purity and maturation\nof CMs following differentiation of hiPSCs. This correlation was validated\nthrough experiments involving different CM-fibroblast mixtures and\nhiPSC-derived CMs with varying efficiencies. Moreover, matured CMs\nexhibited markedly different Raman profiles compared to immature ones,\nindicating the potential of Raman imaging as a tool for assessing\nCM maturation. IV. Conclusions: We discovered that Raman spectroscopy\nof certain components, such as cytochrome <i>c</i> and myoglobin,\ncorrelates with the CM purity and maturation following hiPSC differentiation.\nThe findings of this study highlight the potential of label-free Raman\nmicroscopy as a nondestructive, high-content, and time-efficient method\nfor quality control of hiPSC-derived CMs. This approach could significantly\ncontribute to ensuring the quality and maturity of hiPSC-CMs for various\napplications in drug discovery and regenerative medicine.