Abstract Development of viable cell estimation method without sacrificing proliferation and functions of cells cultured on regenerative biomaterials is essential for regenerative engineering. Cytotoxicity and depletion of resazurin are critical but often overlooked limitations that hindered applications of resazurin in viable cell estimation. The present work found that cytotoxicity and depletion of resazurin depended on cell concentration, resazurin concentration and resazurin incubation time. A simple strategy which only allowed cells to incubate with resazurin during each measurement was developed to eliminate negative effects of resazurin. This strategy was verified by monitoring proliferation of MC3T3-E1 preosteoblasts on poly(d,l-lactic acid) scaffold during a continuous 3D culture process for up to 21 days, comparing the accuracy with MTT assay which is a destructive assay with high sensitivity and accuracy and commonly used in regenerative engineering and comparing viability, proliferation and differentiation functions of MC3T3-E1, which were treated with/without this strategy for nondestructive evaluation. This method showed comparable linearity of standard curve and characteristics of growth curve to MTT assay. No major negative effects of this method on MC3T3-E1 viability and functions were found. Our work highlighted the importance of the concentration and incubation time of resazurin in designing application-specific nondestructive viability assay and would be helpful in improving the implanted medical devices as well as in regenerative engineering.

Flow shear stress controls fundamental behaviors of cells. It can modulate nuclear mechanotransduction through regulating the transport of transcriptional activators such as Yes-associated protein (YAP) from cytoplasm to nucleus. However, the mechanism how flow induces the nucleocytoplasmic transport remains largely unclear. Here we show that flow applied to endothelial cells not only drives polarized actin reorganization but also alters nuclear mechanics, which synergically controls nuclear deformation, resulting in YAP nucleocytoplasmic transport through combination of biological experiments and mechanochemical models. We discovered that unidirectional flow induces simultaneous actin caps formation and nuclear stiffening in a dose and timing-dependent manner, which causes YAP initially entering the nucleus, but then gradually exporting. Additionally, pathological oscillatory flow affects the mechanotransduction by forming slight actin cap and softening nucleus, which sustains YAP nuclear localization. Our work unveils a mechanism of flow-induced nucleocytoplasmic transport, which potentially provides a universal linkage between transcriptional regulation and mechanical stimulation.