Cultivation and recovery of vascular endothelial cells in microchannels of a separable micro-chemical chip

Various micro cell culture systems have recently been developed. However, it is extremely difficult to recover cultured cells from a microchannel because the upper and lower substrates of a microchip are permanently combined. Therefore, we developed a cell culture and recovery system that uses a separable microchip with reversible combining that allows separation between closed and open channels. To realize this system, two problems related to microfluidic control-prevention of leakage and non-invasive recovery of cultured cells from the substrate-must be overcome. In the present study, we used surface chemistry modification to solve both problems. First, octadecyltrimethoxysilane (ODTMS) was utilized to control the Laplace pressure at the liquid/vapor phase interface, such that it was directed toward the microchannels, which suppressed leakage from the slight gap between two substrates. Second, a thermoresponsive polymer poly(N-isopropyl acrylamide) (PNIPAAm) was used to coat the surface of the ODTMS-modified microchannel by UV-mediated photopolymerization. PNIPAAm substrates are well known for controlled cell adhesion/detachment by alteration of temperature. Finally, the ODTMS- and PNIPAAm-modified separable microchips were subjected to patterning, and human arterial endothelial cells (HAECs) were cultured in the resulting microchannels with no leakage. After 96 h of the culture, the HAECs were detached from the microchips by decreasing the temperature and were then recovered from the microchannels. This study is the first to demonstrate the recovery of living cells cultured in a microchannel, and may be useful as a fundamental technique for vascular tissue engineering.