Electrochemical measurement of respiratory activity for evaluation of fibroblast spheroids containing endothelial cell networks

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Vascularized three-dimensional (3D) cultures such as spheroids overcome the problems of insufficient nutrient and oxygen supply and poor cell viability observed with non-vascularized spheroids. However, the capacity of drug penetration may differ with the presence of vascular structures, and needs to be evaluated. Co-culture of endothelial cells (ECs) with other cell types such as fibroblasts is one of the promising methods to engineer vascularized 3D tissue models. At a first step for fabricating vascularized tissues, it is important to prepare EC networks inside the tissue models. Here, we present the first attempt to apply electrochemical measurements of respiratory activity to the fibroblast spheroids containing EC networks, aiming to develop an evaluation technique for vascularized tissue models. The spheroids were exposed to doxorubicin (DOX), an inhibitor of DNA and RNA synthesis, to investigate their drug sensitivity that may differ with the presence of ECs, though rarely studied with regard to respiratory activity. Respiratory activity was measured using two electrochemical devices, a scanning electrochemical microscope (SECM) and a large scale integration (LSI)-based amperometric device. The fibroblast spheroids containing EC networks were constructed with a simple method involving co-culture of normal human dermal fibroblasts (NHDFs) and human umbilical vein endothelial cells (HUVECs) at different concentrations. Fluorescence images showed that HUVECs formed partially networked structure in the fibroblast spheroids. Respiratory activity of the spheroids as measured by SECM varied according to the proportion of HUVECs. In particular, the respiratory activity decreased more in spheroids with a larger proportion of HUVECs after DOX treatment, indicating that drug sensitivity of the spheroids became higher upon incorporation of EC networks. The results with the LSI device also corresponded to those with SECM. Overall, the electrochemical measurements provide meaningful information regarding the EC networks and play an important role in the analysis of 3D cell structures.