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.

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SummaryHigh-purity cardiomyocytes (CMs) derived from human induced pluripotent stem cells (hiPSCs) are promising for drug development and myocardial regeneration. However, most hiPSC-derived CMs morphologically and functionally resemble immature rather than adult CMs, which could hamper their application. Here, we obtained high-quality cardiac tissue-like constructs (CTLCs) by cultivating hiPSC-CMs on low-thickness aligned nanofibers made of biodegradable poly(D,L-lactic-co-glycolic acid) polymer. We show that multilayered and elongated CMs could be organized at high density along aligned nanofibers in a simple one-step seeding process, resulting in upregulated cardiac biomarkers and enhanced cardiac functions. When used for drug assessment, CTLCs were much more robust than the 2D conventional control. We also demonstrated the potential of CTLCs for modeling engraftments in vitro and treating myocardial infarction in vivo. Thus, we established a handy framework for cardiac tissue engineering, which holds high potential for pharmaceutical and clinical applications.

Micro-orifice based cell fusion assures high-yield fusion without compromising the cell viability. This paper examines feasibility of a dielectrophoresis ͑DEP͒ assisted cell trapping method for parallel fusion with a micro-orifice array. The goal is to create viable fusants for studying postfusion cell behavior. We fabricated a microfluidic chip that contained a chamber and partition. The partition divided the chamber into two compartments and it had a number of embedded micro-orifices. The voltage applied to the electrodes located at each compartment generated an electric field distribution concentrating in micro-orifices. Cells introduced into each compartment moved toward the micro-orifice array by manipulation of hydrostatic pressure. DEP assisted trapping was used to keep the cells in micro-orifice and to establish cell to cell contact through orifice. By applying a pulse, cell fusion was initiated to form a neck between cells. The neck passing through the orifice resulted in immobilization of the fused cell pair at micro-orifice. After washing away the unfused cells, the chip was loaded to a microscope with stage top incubator for time lapse imaging of the selected fusants. The viable fusants were successfully generated by fusion of mouse fibroblast cells ͑L929͒. Time lapse observation of the fusants showed that fused cell pairs escaping from micro-orifice became one tetraploid cell. The generated tetraploid cells divided into three daughter cells. The fusants generated with a smaller micro-orifice ͑diameterϳ 2 m͒ were kept immobilized at micro-orifice until cell division phase. After observation of two synchronized cell divisions, the fusant divided into four daughter cells. We conclude that the presented method of cell pairing and fusion is suitable for high-yield generation of viable fusants and furthermore, subsequent study of postfusion phenomena.

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