Influence of chemical fixation process on primary mesenchymal stem cells evidenced by Raman spectroscopy.

In investigation of (patho)physiological processes, cells represent frequently used analyte as an exceptional source of information. However, spectroscopic analysis of live cells is still very seldom in clinics, as well as in research studies. Among others, the reasons are long acquisition time during which autolysis process is activated, necessity of specified technical equipment, and inability to perform analysis in a moment of sample preparation. Hence, an optimal method of preserving cells in the existing state is of extreme importance, having in mind that selection of fixative is cell lineage dependent. In this study, two commonly used chemical fixatives, formaldehyde and methanol, are used for preserving primary mesenchymal stem cells extracted from periodontal ligament, which are valuable cell source for reconstructive dentistry. By means of Raman spectroscopy, cell samples were probed and the impact of these fixatives on their Raman response was analyzed and compared. Different chemical mechanisms are the core processes of formaldehyde and methanol fixation and certain Raman bands are shifted and/or of changed intensity when Raman spectra of cells fixed in that manner are compared. In order to get clearer picture, comprehensive statistical analysis was performed.

Probing primary mesenchymal stem cells differentiation status by micro-Raman spectroscopy.

We have employed micro-Raman spectroscopy to get insight into intrinsic biomolecular profile of individual mesenchymal stem cell isolated from periodontal ligament. Furthermore, these cells were stimulated towards adipogenic, chondrogenic, and osteogenic lineages and their status of differentiation was assessed using micro-Raman spectroscopy. In both cases, glass coverslips were used as substrates, due to their wide availability and cost effectiveness. In all sample groups, the same type of behavior was observed, manifested as changes in Raman spectra: the increase of relative intensity of protein/lipid bands and decrease of nucleic acid bands. Comprehensive statistical analysis in the form of principal component analysis was performed, which revealed noticeable grouping of cells with the similar features. Despite the inhomogeneity of primary stem cells and their differentiated lineages, we demonstrated that micro-Raman spectroscopy is sufficient for distinguishing cells' status, which can be valuable for medical and clinical application.