Biotechnology and bioengineering

Fluorescence optical detection in situ for real-time monitoring of cytochrome P450 enzymatic activity of liver cells in multiple microfluidic devices.

PMID 19575443


We describe an in situ fluorescence optical detection system to demonstrate real-time and non-invasive detection of reaction products in a microfluidic device while under perfusion within a standard incubator. The detection system is designed to be compact and robust for operation inside a mammalian cell culture incubator for quantitative detection of fluorescent signal from microfluidic devices. When compared to a standard plate reader, both systems showed similar biphasic response curves with two linear regions. Such a detection system allows real-time measurements in microfluidic devices with cells without perturbing the culture environment. In a proof-of-concept experiment, the cytochrome P450 1A1/1A2 activity of a hepatoma cell line (HepG2/C3A) was monitored by measuring the enzymatic conversion of ethoxyresorufin to resorufin. The hepatoma cell line was embedded in Matrigel(TM) construct and cultured in a microfluidic device with medium perfusion. The response of the cells, in terms of P450 1A1/1A2 activity, was significantly different in a plate well system and the microfluidic device. Uninduced cells showed almost no activity in the plate assay, while uninduced cells in Matrigel(TM) with perfusion in a microfluidic device showed high activity. Cells in the plate assay showed a significant response to induction with 3-Methylcholanthrene while cells in the microfluidic device did not respond to the inducer. These results demonstrate that the system is a potentially useful method to measure cell response in a microfluidic system.

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Resorufin ethyl ether
Resorufin ethyl ether, suitable for fluorescence, ≥95% (UV)