A technique for monitoring mammalian cell growth and inhibition in situ via Fourier transform infrared spectroscopy.
A single culture of Chinese hamster ovary cells was grown on germanium attenuated total reflectance (ATR) crystals and continuously monitored in situ via ATR/Fourier transform infrared (FT-IR) spectroscopy for approximately 60 h. The cells were seeded into a specially designed flow cell which controlled physiological conditions, flow rate, and addition of growth medium or metabolic inhibitors. Infrared spectra were taken at 20-min intervals until a confluent monolayer was formed. Several strong bands are evident in the spectra which can be generally ascribed to molecular features of cellular components. Cell growth kinetics were measured as a function of infrared band intensity over time and exhibited the normal lag phase, logarithmic growth, and stationary phase on reaching confluence. Spectra of growing cells, normalized to the area under the spectral region 1800-1000 cm-1, were subtracted from reference spectra of confluent cells at 60 h. Difference spectra showed that the largest differences were observed between confluent cells and cells in early growth stages. Differences may reflect cell morphological changes, biochemical activity, and degree of ATR crystal exposure to the bulk medium. ATR/FT-IR spectroscopy of living Chinese hamster ovary cells was also used in a toxicological study to monitor the effects of hydroxyurea, an inhibitor of DNA synthesis. Delayed growth was observed in the cell growth curve of the hydroxyurea-treated cells during the course of treatment as compared to the control culture.
Hutson
T B
TB
National Center for Biomedical Infrared Spectroscopy, Columbus, Ohio 43201-2693.
Mitchell
M L
ML
Keller
J T
JT
Long
D J
DJ
Chang
M J
MJ
D000818:Animals; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D006224:Cricetinae; D003412:Cricetulus; D005260:Female; D005583:Fourier Analysis; D006918:Hydroxyurea / Q000633:toxicity; D007700:Kinetics; D010053:Ovary / Q000166:cytology*; D013055:Spectrophotometry, Infrared / Q000379:methods*
D006918:Hydroxyurea