Reporter cell activity within hydrogel constructs quantified from oxygen-independent bioluminescence.
By providing a three-dimensional (3D) support to cells, hydrogels offer a more relevant in vivo tissue-like environment as compared to two-dimensional cell cultures. Hydrogels can be applied as screening platforms to investigate in 3D the role of biochemical and biophysical cues on cell behaviour using bioluminescent reporter cells. Gradients in oxygen concentration that result from the interplay between molecular transport and cell metabolism can however cause substantial variability in the observed bioluminescent reporter cell activity. To assess the influence of these oxygen gradients on the emitted bioluminescence for various hydrogel geometries, a combined experimental and modelling approach was implemented. We show that the applied model is able to predict oxygen gradient independent bioluminescent intensities which correlate better to the experimentally determined viable cell numbers, as compared to the experimentally measured bioluminescent intensities. By analysis of the bioluminescence reaction dynamics we obtained a quantitative description of cellular oxygen metabolism within the hydrogel, which was validated by direct measurements of oxygen concentration within the hydrogel. Bioluminescence peak intensities can therefore be used as a quantitative measurement of reporter cell activity within a hydrogel, but an unambiguous interpretation of these intensities requires a compensation for the influence of cell-induced oxygen gradients on the luciferase activity.
['Lambrechts|Dennis|D|', 'Roeffaers|Maarten|M|', 'Kerckhofs|Greet|G|', 'Hofkens|Johan|J|', 'Van de Putte|Tom|T|', 'Schrooten|Jan|J|', 'Van Oosterwyck|Hans|H|']
D002452:Cell Count D002470:Cell Survival D002478:Cells, Cultured D036681:Fluorescence Recovery After Photobleaching D017930:Genes, Reporter D057809:HEK293 Cells D006801:Humans D020100:Hydrogels D021621:Imaging, Three-Dimensional D016086:Lentivirus D049449:Luminescence D008163:Luminescent Measurements D008962:Models, Theoretical D010100:Oxygen D012685:Sepharose
Hydrogel Bioluminescence Oxygen Model Luciferase Spatial distribution