A comparative analysis of stem cell differentiation on 2D and 3D substrates using Raman microspectroscopy.

Chondrogenesis is a complex cellular process that involves the transformation of mesenchymal stem cells (MSCs) into chondrocytes, the specialised cells that form cartilage. In recent years, three-dimensional (3D) culture systems have emerged as a promising approach to studying cell behaviour and development in a more physiologically relevant environment compared to traditional two-dimensional (2D) cell culture. The use of these systems provided insights into the molecular mechanisms that regulate chondrogenesis and has the potential to revolutionise the development of new therapies for cartilage repair and regeneration. This study demonstrates the successful application of Raman microspectroscopy (RMS) as a label-free, non-destructive, and sensitive method to monitor the chondrogenic differentiation of bone marrow-derived rat mesenchymal stem cells (rMSCs) in a collagen type I hydrogel, and explores the potential benefits of 3D hydrogels compared to conventional 2D cell culture environments. rMSCs were cultured on 3D substrates for 3 weeks and their differentiation was monitored by measuring the spectral signatures of their subcellular compartments. Additionally, the evolution of high-density micromass cultures was investigated to provide a comprehensive understanding of the process and complex interactions between cells and their surrounding extracellular matrix. For comparison, rMSCs were induced into chondrogenesis in identical medium conditions for 21 days in monolayer culture. Raman spectra showed that rMSCs cultured in a collagen type I hydrogel are able to undergo a distinct chondrogenic differentiation pathway at a significantly higher rate than the 2D culture cells. 3D cultures expressed stronger and more homogeneous chondrogenesis-associated peaks such as collagens, glycosaminoglycans (GAGs), and aggrecan while manifesting changes in proteins and lipidic content. These results suggest that 3D type I collagen hydrogel substrates are promising for in vitro chondrogenesis studies, and that RMS is a valuable tool for monitoring chondrogenesis in 3D environments.