Monitoring the kinetic evolution of mesenchymal stem cell differentiation using Raman microspectroscopy

Raman microspectroscopy (RMS) offers a powerful, non-destructive approach for in situ monitoring of dynamic biochemical processes within cells. However, the ability to reliably data-mine the spectroscopic signatures and their evolution and extract meaningful information can be challenging. Multivariate Curve Resolution-Alternating Least Squares (MCR-ALS) regression analysis is a powerful chemometric technique that can potentially address this challenge by deconvoluting the spectra into individual component spectra, each representing a specific biochemical species, and regressing the solutions against kinetic constraints. In this study, MCR-ALS analysis was performed on spectral data of differentiation process of mesenchymal stem cells into chondrocytes, carried out on two different substrates, collagen 3-dimensional hydrogel and the conventional 2-dimensional culture model at time intervals of 1-7, 14, 21 days. The kinetic evolution of the chondrogenesis was modelled according to a phenomenological rate equation model, in an attempt to describe the biochemical evolution of the cell composition within the process. Moreover, the ability of the algorithm to faithfully extract the correct reaction rates and spectral profiles has been explored. The results indicated that the differentiation process originates in the nucleolar regions, subsequently extending to the nuclear and cytoplasmic compartments and corroborated a more rapid differentiation rate in cell cultures grown on 3D collagen hydrogels compared to 2D substrates. The combination of Raman microspectroscopy and MCR-ALS offers a powerful approach for elucidating the complex mechanisms underlying chondrogenesis and developing innovative strategies for regenerative medicine.