title: Insights into Caco-2 cell culture structure using coherent anti-Stokes Raman scattering (CARS) microscopy.
abstract: We have used coherent anti-Stokes Raman scattering (CARS) microscopy as a novel and rapid, label-free and non-destructive imaging method to gain structural insights into live intestinal epithelial cell cultures used for drug permeability testing. Specifically we have imaged live Caco-2 cells in (bio)pharmaceutically relevant conditions grown on membrane inserts. Imaging conditions were optimized, including evaluation of suitable membrane materials and media solutions, as well as tolerable laser powers for non-destructive imaging of the live cells. Lipid structures, in particular lipid droplets, were imaged within the cells on the insert membranes. The size of the individual lipid droplets increased substantially over the 21-day culturing period up to approximately 10% of the volume of the cross section of individual cells. Variation in lipid content has important implications for intestinal drug permeation testing during drug development but has received limited attention to date due to a lack of suitable analytical techniques. CARS microscopy was shown to be well suited for such analysis with the potential for in situ imaging of the same individual cell-cultures that are used for permeation studies. Overall, the method may be used to provide important information about cell monolayer structure to better understand drug permeation results.
authors.affiliation: Division of Pharmaceutical Chemistry and Technology, Faculty of Pharmacy, FI-00014 University of Helsinki, Finland. Electronic address: jukka.saarinen@helsinki.fi.
authors.forename: Jukka
authors.initials: J
authors.lastname: Saarinen
authors.affiliation: Division of Pharmaceutical Chemistry and Technology, Faculty of Pharmacy, FI-00014 University of Helsinki, Finland.
authors.forename: Erkan
authors.initials: E
authors.lastname: Sözeri
authors.affiliation: Division of Pharmaceutical Chemistry and Technology, Faculty of Pharmacy, FI-00014 University of Helsinki, Finland.
authors.forename: Sara J
authors.initials: SJ
authors.lastname: Fraser-Miller
authors.affiliation: Division of Pharmaceutical Chemistry and Technology, Faculty of Pharmacy, FI-00014 University of Helsinki, Finland.
authors.forename: Leena
authors.initials: L
authors.lastname: Peltonen
authors.affiliation: Division of Pharmaceutical Chemistry and Technology, Faculty of Pharmacy, FI-00014 University of Helsinki, Finland.
authors.forename: Hélder A
authors.initials: HA
authors.lastname: Santos
authors.affiliation: Biomedicum Imaging Unit, Department of Anatomy, Faculty of Medicine, FI-00014 University of Helsinki, Finland.
authors.forename: Antti
authors.initials: A
authors.lastname: Isomäki
authors.affiliation: Division of Pharmaceutical Chemistry and Technology, Faculty of Pharmacy, FI-00014 University of Helsinki, Finland.
authors.forename: Clare J
authors.initials: CJ
authors.lastname: Strachan
mesh_terms: D018938:Caco-2 Cells / Q000166:cytology*; D018929:Cell Culture Techniques; D002470:Cell Survival; D006801:Humans; D008055:Lipids; D013059:Spectrum Analysis, Raman / Q000379:methods
chemical_list: D008055:Lipids
keywords: CARS microscopy; Caco-2 cells; Lipid droplets; Live cell imaging; Non-linear imaging; Raman spectroscopy