From cells to bone : raman microspectroscopy of the mineralization of stromal cells

This thesis presents a body of interdisciplinary research aimed at visualizing bone\ud tissue engineering using microbioreactors and non-invasive and label-free vibrational Raman\ud microspectroscopy. Confocal Raman microspectroscopy is an optical microspectroscopic\ud technique enabling spatially resolved chemical imaging and analysis. In this thesis this\ud approach was used to monitor the temporal development of in vitro bone formation, that is,\ud from proliferation of human bone marrow derived stromal cells, their differentiation towards\ud osteogenic precursors, till early and late stages of mineralized tissue formation. The results\ud yield deeper insights into, and understanding of, in vitro formation of mineralized tissue from\ud stromal cells derived from human bone marrow that are consistent with the characteristics of\ud in vivo bone formation

The intensity of the 1602 cm−1 band in human cells is related to mitochondrial activity

We report a Raman band at 1602 cm−1 in the spectra of human cells, which previously had only been observed in mitochondria of yeast cells. This band, which has not yet been assigned to a particular molecular species, was found to occur in HeLa cells, peripheral blood lymphocytes, human mesenchymal stem cells and bovine chondrocytes. The band is proposed as an indicator of the activity of mitochondria in cells. Cells were cultured with and without serum or temporarily deprived of serum. The band can be observed for all these variations in cell culture methodology. The band intensity decreases under the influence of an increase of the calcium ion concentration in the surrounding medium

Microbioreactors for Raman microscopy of stromal cell differentiation

We present the development of microbioreactors with a sensitive and accurate optical coupling to a confocal Raman microspectrometer. We show that such devices enable in situ and in vitro investigation of cell cultures for tissue engineering by chemically sensitive Raman spectroscopic imaging techniques. The optical resolution of the Raman microspectrometer allows recognition and chemical analysis of subcellular features. Human bone marrow stromal cells (hBMSCs) have been followed after seeding through a phase of early proliferation until typically 21 days later, well after the cells have differentiated to osteoblasts. Long-term perfusion of cells in the dynamic culture conditions was shown to be compatible with experimental optical demands and off-line optical analysis. We show that Raman optical analysis of cells and cellular differentiation in microbioreactors is feasible down to the level of subcellular organelles during development. We conclude that microbioreactors combined with Raman microspectroscopy are a valuable tool to study hBMSC proliferation, differentiation, and development into tissues under in situ and in vitro conditions