Page 107 - Vibrational Spectroscopic Imaging for Biomedical Applications

Sample Pr eparation of Cells and T issue 83

for FTIR experiments or quartz slides for Raman experiments, by
incubation for 24 hours at 4ºC. Laminin and fibronectin were coated
onto these substrates by incubation for 4 hours and 40 minutes,
respectively, at room temperature. For the fibronectin- and laminin-
coated slides, excess solution was aspirated from the substrates and
washed in PBS prior to cell deposition. Whereas, for the gelatin-coated
slide, excess solution was aspirated and cells were deposited for culture
without prior washing in PBS. Fluorescence assays were conducted at
3 days postseeding as well as fixation using 4 percent formalin (in PBS)
with water rinse for FTIR and Raman investigations.
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Meade et al. observed through fluorescence assays that cellular
proliferation, viability as well as protein content were down-regulated
when cells were grown on uncoated quartz compared with uncoated
MirrIR substrates. However, increases in proliferation and viability
were more pronounced when cells were grown on coated quartz than
grown on coated MirrIR substrates. Additionally, it was found that
quartz coated with all three ECM coatings generated significantly
enhanced proliferation compared to the control (uncoated quartz).
However, this was not the case for MirrIR, which resulted in a sig-
nificant increase in proliferation only for the laminin-coated slide.
Viability was significantly increased when cells were grown on lam-
inin- and gelatin-coated quartz, whereas for MirrIR substrate, viabil-
ity was only significantly increased when this was coated with gelatin.
The authors suggest that gelatin provides a coating with similar pro-
liferation effects on quartz and MirrIR and increases viability, which
is desirable for long-term cultures.
FTIR and Raman spectroscopic analyses of coated slides demon-
strate that the gelatin coating did not give rise to sufficiently high sig-
nals to significantly influence FTIR or Raman spectra of the cells cultured
upon them. First derivative FTIR spectra and Raman spectra of cells on
gelatin, fibronectin and laminin demonstrated spectral changes on each
of these substrates that were associated with nucleic acid, lipid, and
protein expression. Raman spectra provided further insight and relative
quantification, since it was found that coatings that promoted prolifera-
tion gave rise to increases in spectral regions associated with DNA,
RNA, and proteins, with a decrease in lipids. This has been attributed to
an increase in the sustained production of signaling proteins, as a result
of integrin binding to the coating, that promotes cellular proliferation.
Supporting this, the authors also found through FTIR that the ratio of
protein (sum of integral absorbencies corresponding to the amide I, II,
−1
and III bands) to lipid (integral absorbance 1370 to 1400 cm ) gave rise
to values that could be significantly correlated with an increase in pro-
liferation (as measured by fluorescence spectroscopy).
The experimental setup described above consisted of thin layers
of ECM that were barely detectable in the FTIR or Raman spectrum;
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however, Lee et al. studied prostate cancer cells that had been cultured
onto relatively thicker layers of ECM. In their investigation, Matrigel