Assessment of liver function in primary cultures of hepatocytes using diethoxy (5,6) chloromethylfluorescein and confocal laser scanning microscopy.
A method is presented which can be used to assess the function of hepatocytes in complex culture configurations without disrupting the integrity of the cell environment. It utilises a fluorescent probe for cytochrome P450 dependent mixed function oxidase (MFO) activity diethoxy (5,6) chloromethylfluorescein, and confocal laser scanning microscopy. The MFO activity of individual cells in primary cultures of intact hepatocytes can be detected in situ, and quantified by image analysis. This may be a valuable means of monitoring the effect of culture conditions on the function of bioartificial liver devices, and could be used to assess the need for effective oxygenation of cells, the influence of shear stress and of exposure to patient serum during clinical use.
Bioengineering Unit, Strathclyde University, Wolfson Centre, Glasgow, UK.
K
K
Anderson
R
R
Wilkinson
M H
MH
Grant
D000818:Animals; D002478:Cells, Cultured; D003577:Cytochrome P-450 Enzyme System / Q000032:analysis; D005452:Fluoresceins; D005456:Fluorescent Dyes; D008099:Liver / Q000201:enzymology*; D008297:Male; D018613:Microscopy, Confocal; D006899:Mixed Function Oxygenases / Q000032:analysis; D051381:Rats; D017207:Rats, Sprague-Dawley
C115471:5,6-diethoxychloromethylfluorescein; D005452:Fluoresceins; D005456:Fluorescent Dyes; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases