Tissue Engineering Part C, Methods vol:19 issue:9 pages:720-729
Bone Tissue Engineering (TE) aims to develop reproducible and predictive three-dimensional (3D) TE constructs, defined as cell-seeded scaffolds produced by a controlled in vitro process, to heal or replace damaged and non-functional bone. To control and assure the quality of the bone TE constructs, a prerequisite for regulatory authorization, there is a need to develop non-invasive analysis techniques to evaluate TE constructs and to monitor their behavior in real time during in vitro culturing. Most analysis techniques, however, are limited to destructive end-point analyses. This study investigates the use of the non-toxic alamarBlue® (AB) reagent, which is an indicator for metabolic cell activity, for monitoring the cellularity of 3D TE constructs in vitro as part of a bioreactor culturing processes. Within the field of TE, bioreactors have a huge potential in the translation of TE concepts to the clinic. Hence, the use of the AB reagent was not only evaluated in static cultures, but also in dynamic cultures in a perfusion bioreactor set-up. Hereto, the AB assay was successfully integrated in the bioreactor-driven TE construct culture process in a non-invasive way. The obtained results indicate a linear correlation between the overall metabolic activity and the total DNA content of a scaffold upon seeding as well as during the initial stages of cell proliferation. This makes the AB reagent a powerful tool to follow-up bone TE constructs in real-time during static as well as dynamic 3D cultures. Hence the AB reagent can be successfully used to monitor and predict cell confluence in a growing 3D TE construct.