Efficiency of doxorubicin handling by isolated hepatocytes is a valuable indicator for restored cell function

Pig liver is a possible source of hepatocytes for extracorporeal bio-artifi cial liver devices. In order to evaluate recovered hepatocyte function foll owing enzymatic isolation, we developed a cytochemical method that is based on the capacity of hepatocytes to sequester the anthracycline antitumour d rug doxorubicin within intracellular acidic compartments. Doxorubicin is a naturally fluorescent molecule. Thus, the process of drug concentration wit hin hepatocytes can be visualized in living conditions by fluorescence micr oscopy. Porcine hepatocytes harvested from heart-beating donors were grown either as isolated cell suspensions or as tissue monolayers. Immediately af ter isolation and at fixed culture times, cells were incubated with 0.1 mM doxorubicin in Hanks' balanced salt solution for 10 min at 37 degreesC in 5 % CO2-humidified atmosphere and observed by fluorescence microscopy. Parall el electron microscopy was performed to compare fluorescence data with gene ral cell morphology. To monitor lysosomal acidification capacity, the fluor escent pH-sensitive vital dye LysoSensor-Blue was used. Doxorubicin fluores cence showed different patterns of nuclear and cytoplasmic staining, accord ing to the time allowed for cell recovery and the culture method. In partic ular, cytoplasmic fluorescence changed from a diffuse staining, that could be observed after cell isolation and in hepatocyte suspensions, to a puncta te perinuclear and pericanalicular fluorescence detectable in fully recover ed hepatocyte monolayers. This study indicates that the 'doxorubicin-fluore scence test' may be considered a simple and rapid procedure for assessing h epatocyte functional condition. It may provide valuable and 'real time' gui delines for judging the correct way these cells are to be collected, preser ved and utilized for clinical purposes.