title: Reporter cell activity within hydrogel constructs quantified from oxygen-independent bioluminescence.
abstract: 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.
authors: Department of Materials Engineering, KU Leuven, Kasteelpark Arenberg 44 - Box 2450, 3001 Leuven, Belgium; Prometheus, Division of Skeletal Tissue Engineering Leuven, KU Leuven, Herestraat 49 - Box 813, 3000 Leuven, Belgium.
authors: Dennis
authors: D
authors: Lambrechts
authors: Center for Surface Chemistry and Catalysis, KU Leuven, Kasteelpark Arenberg 23, 3001 Leuven, Belgium.
authors: Maarten
authors: M
authors: Roeffaers
authors: Department of Materials Engineering, KU Leuven, Kasteelpark Arenberg 44 - Box 2450, 3001 Leuven, Belgium; Prometheus, Division of Skeletal Tissue Engineering Leuven, KU Leuven, Herestraat 49 - Box 813, 3000 Leuven, Belgium.
authors: Greet
authors: G
authors: Kerckhofs
authors: Molecular Imaging and Photonics, KU Leuven, Celestijnenlaan 200F, 3001 Leuven, Belgium.
authors: Johan
authors: J
authors: Hofkens
authors: TiGenix NV, Haasrode Researchpark 1724, Romeinse Straat 12 box 2, 3001 Leuven, Belgium.
authors: Tom
authors: T
authors: Van de Putte
authors: Department of Materials Engineering, KU Leuven, Kasteelpark Arenberg 44 - Box 2450, 3001 Leuven, Belgium; Prometheus, Division of Skeletal Tissue Engineering Leuven, KU Leuven, Herestraat 49 - Box 813, 3000 Leuven, Belgium. Electronic address: Jan.Schrooten@kuleuven.be.
authors: Jan
authors: J
authors: Schrooten
authors: Prometheus, Division of Skeletal Tissue Engineering Leuven, KU Leuven, Herestraat 49 - Box 813, 3000 Leuven, Belgium; Biomechanics Section, KU Leuven, Celestijnenlaan 300C - Box 2419, 3001 Leuven, Belgium. Electronic address: Hans.VanOosterwyck@kuleuven.be.
authors: Hans
authors: H
authors: Van Oosterwyck
mesh_terms: D002452:Cell Count; D002470:Cell Survival; D002478:Cells, Cultured; D036681:Fluorescence Recovery After Photobleaching; D017930:Genes, Reporter; D057809:HEK293 Cells; D006801:Humans; D020100:Hydrogels / Q000737:chemistry*; D021621:Imaging, Three-Dimensional; D016086:Lentivirus / Q000235:genetics; D049449:Luminescence; D008163:Luminescent Measurements / Q000379:methods*; D008962:Models, Theoretical; D010100:Oxygen / Q000737:chemistry*; D012685:Sepharose
chemical_list: D020100:Hydrogels; D012685:Sepharose; D010100:Oxygen
keywords: Bioluminescence; Hydrogel; Luciferase; Model; Oxygen; Spatial distribution