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In the field of tissue engineering andregenerative medicine, there is a demand for construction of three-dimensional(3D) organs such as liver, pancreas, and kidney. Although recent advances intissue engineering allowed us to construct two-dimensional tissues such as skinand cornea, it is still challenging to construct 3D organs. Since these vitalorgans are complex in structure and contain abundant capillary networks, it isdifficult to construct these organs in vitro. In particular, construction of capillarynetworks and their integration to epithelial tissues are important for 3Dtissue engineering. Therefore, we have been focusing on construction capillarynetworks. The aim of this study is to investigate the mechanism how we canconstruct functional microvascular networks by the co-culture of endothelialcells and mesenchymal stem cells while we utilize a microfluidic device tocontrol culture microenvironments. Microfluidicdevices used in this study were made by soft lithography. Our microfluidicdevice is made of silicone rubber (polydimethylsiloxane) and a cover glass. Thedevice has two parallel microfluidic channels and an intervening hydrogelregion, which allowed us to control cellular distribution and interstitial flowconditions. Using this microfluidic device, we cultured human umbilical veinendothelial cells (HUVEC) and mesenchymal stem cells (MSC). Phase-contrast and confocalfluorescent microscopy methods were used to investigate the process ofcapillary formation. HUVEC,which were seeded into a microfluidic channel, attached on the sidewall made ofhydrogel and started to penetrate into the gel for constructing vascularsprouts. These vascular sprouts extended into the gel and finally developedinto branching capillary networks. However, when HUVEC were cultured alone,capillary networks were not able to be maintained in long-term culture. On theother hand, when HUVEC were culture with MSC, MSC migrated into gel region, andsome cells attached to HUVEC differentiated into pericytes. After MSC attachedto HUVEC, HUVEC started to form vascular sprouts and finally developed into branchingcapillary networks, which were stable for more than 3 weeks. Confocalmicroscopy revealed that these capillary networks contained continuous lumens,which were partially wrapped by pericytes, suggesting that pericytes playimportant roles in stabilization of constructed capillary networks in vitro. Inaddition, the effect of interstitial flow on the construction of microvascularnetworks was investigated by applying a series of interstitial flow magnitude.The results revealed that capillary morphogenesis was enhanced with increasinginterstitial flow magnitude. In conclusion, HUVEC-MSC interactions andinterstitial flow conditions play important roles in regulating capillaryformation. Control of these culture conditions allows us to constructlong-lasting functional microvascular networks.