Creating vascular networks in tissues is crucial for tissue engineering. Although recent studies have demonstrated the formation of vessel-like structures in a tissue model, long-term culture is still challenging due to the lack of active perfusion in vascular networks. Here, we present a method to create a three-dimensional cellular spheroid with a perfusable vascular network in a microfluidic device. By the definition of the cellular interaction between human lung fibroblasts (hLFs) in a spheroid and human umbilical vein endothelial cells (HUVECs) in microchannels, angiogenic sprouts were induced from microchannels toward the spheroid; the sprouts reached the vessel-like structures in a spheroid to form a continuous lumen. We demonstrated that the vascular network could administer biological substances to the interior of the spheroid. As cell density in the spheroid is similar to that of a tissue, the perfusable vasculature model opens up new possibilities for a long-term tissue culture in vitro.

We report a microfluidic system that physically separates nuclear RNA (nucRNA) and cytoplasmic RNA (cytRNA) from a single cell and enables single-cell integrated nucRNA and cytRNA-sequencing (SINC-seq). SINC-seq constructs two individual RNA-seq libraries, nucRNA and cytRNA, per cell, quantifies gene expression in the subcellular compartments, and combines them to create novel single-cell RNA-seq data. Leveraging SINC-seq, we discover distinct natures of correlation among cytRNA and nucRNA that reflect the transient physiological state of single cells. These data provide unique insights into the regulatory network of messenger RNA from the nucleus toward the cytoplasm at the single-cell level.

Scanning electrochemical microscopy (SECM) is a powerful method for evaluating three-dimensional culture models. In this study, we investigated the oxygen consumption rate of tumor spheroids during the formation of necrotic-like core using SECM.

Current in vitro 3D culture models lack a vascular system to transport oxygen and nutrients, as well as cells, which is essential to maintain cellular viability and functions. Here, we describe a microfluidic method to generate a perfusable vascular network that can form inside 3D multicellular spheroids and functionally connect to microchannels. Multicellular spheroids containing endothelial cells and lung fibroblasts were embedded within a hydrogel inside a microchannel, and then, endothelial cells were seeded into both sides of the hydrogel so that angiogenic sprouts from the cell spheroids and the microchannels were anastomosed to form a 3D vascular network. Solution containing cells and reagents can be perfused inside the cell spheroids through the vascular network by injecting it into a microchannel. This method can be used to study cancer cell migration towards 3D co-culture spheroids through a vascular network. We recapitulated a bone-like microenvironment by culturing multicellular spheroids containing osteo-differentiated mesenchymal stem cells (MSCs), as well as endothelial cells, and fibroblasts in the device. After the formation of vascularized spheroids, breast cancer cells were injected into a microchannel connected to a vascular network and cultured for 7 days on-chip to monitor cellular migration. We demonstrated that migration rates of the breast cancer cells towards multicellular spheroids via blood vessels were significantly higher in the bone-like microenvironment compared with the microenvironment formed by undifferentiated MSCs. These findings demonstrate the potential value of the 3D vascularized spheroids-on-a-chip for modeling in vivo-like cellular microenvironments, drug delivery through blood vessels, and cellular interactions through a vascular network.