Fluorescence optical detection in situ for real-time monitoring of cytochrome P450 enzymatic activity of liver cells in multiple microfluidic devices.
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.
Sung
Jong Hwan
JH
School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, New York, USA.
Choi
Jong-ryul
JR
Kim
Donghyun
D
Shuler
Michael L
ML
D018929:Cell Culture Techniques; D045744:Cell Line, Tumor; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D005453:Fluorescence; D022781:Hepatocytes / Q000201:enzymology*; D006801:Humans; D046210:Microfluidic Analytical Techniques; D010078:Oxazines / Q000378:metabolism
D010078:Oxazines; C007649:ethoxyresorufin; C014180:resorufin; D003577:Cytochrome P-450 Enzyme System