A model-driven pipeline for measuring oxygen consumption rates in cell-laden microspheres

The rate of oxygen consumption in in vitro systems is widely considered as a criterion for evaluating their “goodness” in terms of functional similarity to their in vivo counterparts, a crucial step toward the establishment of translatable—or physiologically relevant—models. However, current metrics for the characterization of oxygen consumption are ambiguously defined and often come from inappropriate experimental approximations. We developed a model-driven pipeline, which integrates biofabrication, oxygen micro-sensing technologies, and computational methods to measure the overall and cell-specific oxygen consumption rates of hepatocyte-laden hydrogel microspheres of different sizes. The experiments are based on direct evaluation of spatial concentration gradients inside the microspheres; hence, the method overcomes some of the limitations related to bulk-level measurements, typical of current systems. Our results suggest that hepatic microspheres with masses between 9.0 and 21.3 mg do not follow allometric scaling in oxygen consumption rate. Furthermore, we demonstrate that our pipeline suffers from negligible instrumental noise (≤ 0.068% of the median cell-specific oxygen consumption rate) in comparison with the variability of oxygen uptake by individual cell constructs, which was measured as higher than the 72.3% of the corresponding median value. The pipeline and methods developed can be easily generalized to any tissue construct of spheroidal shape (e.g., high-cell density spheroids, organoids) and are a powerful toolkit for rigorous and quantitative assessments of metabolic processes, and for capturing their variations and associated emergent properties (such as size-related scaling) in vitro, as well as for the optimization of pathophysiological tissue modeling.