Three dimensional collagen gels as a cell culture matrix for the study of live cells by Raman spectroscopy

Abstract

Three dimensional collagen gels are evaluated as matrices for the study of live cells by Raman spectroscopy. The study is conducted on a human lung adenocarcinoma (A549) and a spontaneously immortalized human epithelial keratinocyte (HaCaT) cell line. It is demonstrated, using the Alamar Blue assay, that both cell models exhibit enhanced viability in collagen matrices compared to quartz substrates, commonly used for Raman spectroscopy. Using principal component analysis, it is shown that the Raman spectral analysis of cells in collagen matrices are minimally contaminated by substrate contributions and the cell to cell spectral variations are greatly reduced compared to those measured on quartz substrates. Furthermore, the spectral measurements are seen to have little contribution from the cell culture medium, implying that cultures can be kept viable over prolonged measurement or mapping procedures.

Imaging live cells grown on a three dimensional collagen matrix using Raman microspectroscopy

“…The feasibility of recording Raman spectra from cells in the presence of the culture medium without interference to the spectra has been reported 22 . However, the main limitations concerning the collagen gels seem to be the luminosity and the contrast when observing the cells by white light in transmission.…”

“…Examples of spectra corresponding to the background and cellular spectrum are given in the figure 2A1 and 2A2. The signal of the collagen doesn't contain any peaks and represents the contribution of the water present in the gel and in the aqueous solution used to keep the cells alive 22 . Before removal of the background signal, the spectra were subjected to a light smoothing (Savitzky-Golay, 13 pts).…”

“…As measured using the Alamar Blue cytotoxicity assay, in comparison to uncoated quartz, a human lung cell line exhibited a 39% increase in viability in collagen gels whereas as dermal cell line seemed to be more affected by the different substrates and a 285% increase in viability was observed. Furthermore, after polymerization, the density of the collagen is too low to give any contribution in the spectra recorded 22 . Also no contribution from the medium used for the cell culture is observable in the spectra recorded which allows the cells to be kept alive for prolonged periods and spectroscopically monitored, effectively in real time.…”

Improved protocols for vibrational spectroscopic analysis of body fluids

“…Whereas dry samples are often required for FTIR analysis of tissue sections, Raman microspectroscopy is perfectly compatible with water immersion measurement which enhances the signal to background ratio and the collection of higher quality data [36,37]. The weak contribution from water offers the possibility to study cells in an aqueous environment and thus to keep them alive for the duration of the measurement [2,31,38], whereas the strong absorption from water means that such measurements in infrared can only be performed with high brightness sources such as synchrotrons [39].…”

Spectropathology for the next generation: Quo vadis?

“…42 It is important to note that, although the signal is small, water does contribute to the underlying background and careful preprocessing of the data is essential, remembering that water is also a constituent intracellular component.…”