Whole organ bioengineering by recellularization of decellularized matrix is a new promising technology, which aims to address the problem of organs shortage in transplantation. Because bioengineered organ is maintained in culture as a whole rather than cell suspension, one may ask about daily cell viability assessment in these settings. Harald Ott’s group from Harvard, recently proposed to use colorimetric Resazurin assay for testing cell viability of bioengineered organs ex vivo.
A reliable colorimetric or fluorometric assay for repetitive quantification of viable cell numbers in large-size, three-dimensional bioengineered organ constructs needs to meet the following criteria. First, the diffusion of substrate dye and its metabolized product into and out of bioengineered constructs needs to be efficient, which ensures that the sampling from culture medium is representative of the composition of substrate dye and its metabolized product inside the constructs. Second, the reagents need to have minimal cytotoxicity during the assay period and need to be efficiently washed out after a measurement, thus enabling repetitive measurement during the entire culture period of bioengineered organ constructs.
Resazurin is metabolized by live cells and can be detected by fluorescent product-metabolite in the culture medium. Reduction of resazurin with time is proportional to cell number changes. For assay validation, it should be compared with standard direct cell counting after tissue dissociation. For 3D tissue engineered constructs, the authors proposed resazurin reduction perfusion assay:
… first, the resazurin-based PrestoBlue reagent with low cytotoxicity, efficient metabolism and diffusion allows repetitive cell number quantification; second, resazurin delivery by means of perfusion through the organs’ own vascular bed greatly decreases diffusion distances allowing efficient and homogeneous distribution of the colorimetric and fluorometric reagent throughout tissue-engineered constructs.
Perfusion allowed good equilibration of dye in the organ. Researchers compared this method on 2 different endothelial cell types, seeded on rat decell lung biomatrix.
Viability estimates in the study were very close to histological assessment. The authors think that organ perfusion method could be used for other reagents. Overall, this method allows long-term precise daily estimation of cell viability in 3D cultured tissues/ organs, including large volume bioreactors.