Vibrational spectroscopic monitoring and biochemical analysis of pericellular matrix formation and maturation in a 3-dimensional chondrocyte culture model.

Isolated and monolayer expanded chondrocytes are not the ideal cell form to produce a cartilage matrix. In articular cartilage, each chondrocyte is surrounded by a 2-4 µm thick collagen VI-rich pericellular matrix (PCM) forming a chondron. Freshly extracted chondrons form a more cartilage-like extracellular matrix (ECM) than chondrocytes and their surrounding PCM is thought to maintain the chondrocyte phenotype. To regenerate articular cartilage, preserving and/or regenerating a functional PCM is essential. In this study, a highly biomimicking hyaluronic acid (HA) hydrogel was used as a 3-dimensional system to culture freshly isolated bovine chondrons (with an intact PCM) and chondrocytes (without a PCM) for up to 21 days. We assessed the HA hydrogel's capacity to maintain and potentially re-generate PCM formation by both biochemical and immunological analyses of the key components of the PCM. For the first time, synchrotron based Fourier transform infrared (SR-FTIR) microspectroscopy was utilised to reveal the dynamic process of PCM re-generation. At day 1, highly specific collagen VI staining was visible within chondron containing HA hydrogels. In contrast, collagen VI was absent at day 1 but punctate, focal staining increased during the culture period of chondrocyte containing HA hydrogels. Chondron containing HA hydrogels produced more collagen II and GAGs than the chondrocyte containing HA hydrogels. Principal component analysis (PCA) of spectra in fingerprint regions of the chondrocyte-containing constructs at day 7, 14 and 21 culturing showed clear spectral differences. The clusters of day 14 and day 21 samples were closer to the chondron samples, while the day 7 samples were closer to chondrocytes. PCA scores in the lipid region revealed no major differences between chondrocyte and chondron samples, but showed that the cultured chondrocyte samples at day 7, day 14 and day 21 clustered together. These data would indicate that SR-FTIR microspectroscopy can help to better understand the PCM formation and maturation in tissue engineered models, which involves subtle changes in collagen and aggrecan.

Fourier transform infrared spectra of cells on glass coverslips. A further step in spectral pathology.

Over the last few years, great effort has been placed on developing Fourier Transform Infrared (FTIR) microspectroscopy as a tool to help in the histopathological diagnosis of cancer. The ever increasing workload in pathology departments is calling for a technique that could identify the presence of cancer cells in cytology and tissue samples in an objective, fast and automated way. However, pathologists use glass slides which absorb infrared (IR) radiation thus removing important mid-IR spectral data in the fingerprint region (proteins, DNA, RNA; 1800 cm-1 to 900 cm-1). To this purpose, we hypothesised whether using thinner glass slides, i.e., glass coverslips, would allow us to obtain spectral data not only from the lipid region (3100 cm-1 to 2700 cm-1) but also from the fingerprint region. To this purpose, we studied peripheral blood mononuclear cells (PBMC), a leukaemia cell line (K562) and a lung cancer cell line (CALU-1). Cells were placed on DAKO coverslips and their FTIR spectra obtained at MIRAS beamline, Alba synchrotron light source (Barcelona, Catalonia). The data presented here not only shows for the first time that it is possible to obtain spectral data from most of the amide I region (1800 cm-1 to 1570 cm-1) of cells placed on glass coverslips but more important, principal component analysis was able to separate between the three types of cells for both the lipid and the amide I regions. The methodology here described is a further step in the application of FTIR microspectroscopy in histopathology departments.

Effects of nilotinib on leukaemia cells using vibrational microspectroscopy and cell cloning.

Over the last few years, both synchrotron-based FTIR (S-FTIR) and Raman microspectroscopies have helped to better understand the effects of drugs on cancer cells. However, cancer is a mixture of cells with different sensitivity/resistance to drugs. Furthermore, the effects of drugs on cells produce both chemical and morphological changes, the latter could affect the spectra of cells incubated with drugs. Here, we successfully cloned sensitive and resistant leukaemia cells to nilotinib, a drug used in the management of leukaemia. This allowed both the study of a more uniform population and the study of sensitive and resistant cells prior to the addition of the drug with both S-FTIR and Raman microspectroscopies. The incubation with nilotinib produced changes in the S-FTIR and Raman spectra of both sensitive and resistant clones to nilotinib. Principal component analysis was able to distinguish between cells incubated in the absence or presence of the drug, even in the case of resistant clones. The latter would confirm that the spectral differences between the so-called resistant clonal cells prior to and after adding a drug might reside on those more or less sensitive cells that have been able to remain alive when they were collected to be studied with S-FTIR or Raman microspectroscopies. The data presented here indicate that the methodology of cell cloning can be applied to different types of malignant cells. This should facilitate the identification of spectral biomarkers of sensitivity/resistance to drugs. The next step would be a better assessment of sensitivity/resistance of leukaemia cells from patients which could guide clinicians to better tailor treatments to each individual patient.