Abstract
Bioluminescence imaging assays have become a widely integrated technique to quantify effectiveness of cell-based therapies by monitoring fate and survival of transplanted cells. To date these assays are still largely qualitative and often erroneous due to the complexity and dynamics of local micro-environments (niches) in which the cells reside. Here, we report, using a combined experimental and computational approach, on oxygen that besides being a critical niche component responsible for cellular energy metabolism and cell-fate commitment, also serves a primary role in regulating bioluminescent light kinetics. We demonstrate the potential of an oxygen dependent Michaelis-Menten relation in quantifying intrinsic bioluminescence intensities by resolving cell-associated oxygen gradients from bioluminescent light that is emitted from three-dimensional (3D) cell-seeded hydrogels. Furthermore, the experimental and computational data indicate a strong causal relation of oxygen concentration with emitted bioluminescence intensities. Altogether our approach demonstrates the importance of oxygen to evolve towards quantitative bioluminescence and holds great potential for future microscale measurement of oxygen tension in an easily accessible manner.
Highlights
In situ studies on the mechanisms of cell fate regulation in local microenvironments has gained considerable interest in the development of cell based therapies for disease and regeneration. These studies are very often complemented with bioluminescence imaging assays that yield valuable information on cell fate and behavior in a dynamic microenvironment
Bioluminescence imaging relies on the activity of luciferase enzymes that act as catalyst for the conversion of luciferin to oxyluciferin, which is accompanied by the release of a photon [5]
Previous studies have elucidated the effects of oxygen concentration on the emitted bioluminescence intensity [9,10], we show here how a mathematically validated model aids in resolving the oxygen dependent influences and how this model can be used to obtain quantitative measurements of the intrinsic bioluminescence intensity
Summary
In situ studies on the mechanisms of cell fate regulation in local microenvironments (niches) has gained considerable interest in the development of cell based therapies for disease and regeneration. Oxygen dependent changes in initial bioluminescence reaction kinetics were determined from dynamic time point measurements of luciferase activity with varying luciferin concentrations. Cells exposed to an initial luciferin substrate concentration of 470 mM presented a bi-exponential decay in their luciferase activity (Fig. 2E,F) [23].
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