Glycosaminoglycan profiling in different cell types using infrared spectroscopy and imaging

Abstract

We recently identified vibrational spectroscopic markers characteristic of standard glycosaminoglycan (GAG) molecules. The aims of the present work were to further this investigation to more complex biological systems and to characterize, via their spectral profiles, cell types with different capacities for GAG synthesis. After recording spectral information from individual GAG standards (hyaluronic acid, chondroitin sulfate, dermatan sulfate, heparan sulfate) and GAG-GAG mixtures, GAG-defective mutant Chinese hamster ovary (CHO)-745 cells, wild-type CHO cells, and chondrocytes were analyzed as suspensions by high-throughput infrared spectroscopy and as single isolated cells by infrared imaging. Spectral data were processed and interpreted by exploratory unsupervised chemometric methods based on hierarchical cluster analysis and principal component analysis. Our results showed that the spectral information obtained was discriminant enough to clearly delineate between the different cell types both at the cell suspension and single-cell levels. The abilities of the technique are to perform spectral profiling and to identify single cells with different potentials to synthesize GAGs. Infrared microspectroscopy/imaging could therefore be developed for cell screening purposes and further for identifying GAG molecules in normal tissues during physiological conditions (aging, healing process) and numerous pathological states (arthritis, cancer).

Implementation of infrared and Raman modalities for glycosaminoglycan characterization in complex systems

“…Brézillon and collaborators [54] applied IR microspectroscopy and imaging to identify in a first step the spectroscopic signatures of GAG standards, GAG-Collagen mixtures, and in a second step cells expressing different levels of GAGs, both in suspensions and in isolated single cells. Cartilage chondrocytes are the cells with the highest capacity for GAGs synthesis.…”

“…, HA, HEP, HS, C4S, C6S, DS and GAG-collagen mixtures. We next investigated cellular IR spectroscopy of CHO-745, CHO-WT, chondrocytes [54] and SK-MEL-28, dermal fibroblast (unpublished data) in suspension and performed IR imaging of single cells. Spectral data were analysed by HCA chemometric method in order to characterize these different cell types exhibiting from low to high levels of GAG synthesis.…”

“…Spectral data were analysed by HCA chemometric method in order to characterize these different cell types exhibiting from low to high levels of GAG synthesis. We identified the infrared spectral signatures of standard GAGs and differentiated cell types based on their capacity of GAG synthesis [54]. …”

“…Spectroscopic markers characteristic of reference GAG molecules have been identified previously based on their molecular structures by Raman and infrared (IR) microspectroscopies [52, 53]. These techniques are sensitive enough to descriminate different cell types according to their capacity to synthetise GAGs [54]. Both IR and Raman microspectroscopies are highly sensitive to the structure, composition, and environment of the molecules constituting the studied specimen.…”

“…These studies concerned only a particular type of GAGs or digested molecules. The first IR spectral signatures of GAG in complex biological systems like cells were also recently demonstrated [54]. However, IR microspectroscopy is limited in terms of spatial resolution and also requires sample drying.…”