• Xenotransplantation 01/1997; 4(4):267-274. · 2.57 Impact Factor
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    ABSTRACT: Cryopreservation, the freezing of hepatocytes in liquid nitrogen for storage, has been investigated for many years, as a method of long-term storage for hepatocytes. Unfortunately an agreed acceptable protocol has been elusive, in part due to the susceptibility of hepatocytes to the freeze thaw process involved. A method for long-term storage (months, possibly years) of human hepatocytes, in particular, is desirable for the development of a clinically applicable bioartificial liver, hepatocyte transplantation and for pharmacotoxicological research. The sources of human liver tissue from which hepatocytes can be derived are limited. Many groups have modified and improved the process of cryopreservation and many new techniques have been published, including the incorporation of such cryopreserved cells in clinically based studies. Further evaluation is still required to develop a universally acceptable protocol. This article reviews the difficulties involved in cryopreserving hepatocytes for banking and examines recent technical advances within this field.
    Cell and Tissue Banking 02/2003; 4(1):3-15. · 1.17 Impact Factor
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    ABSTRACT: Pig liver is a possible source of hepatocytes for extracorporeal bio-artificial liver devices. In order to evaluate recovered hepatocyte function following enzymatic isolation, we developed a cytochemical method that is based on the capacity of hepatocytes to sequester the anthracycline antitumour drug doxorubicin within intracellular acidic compartments. Doxorubicin is a naturally fluorescent molecule. Thus, the process of drug concentration within hepatocytes can be visualized in living conditions by fluorescence microscopy. Porcine hepatocytes harvested from heart-beating donors were grown either as isolated cell suspensions or as tissue monolayers. Immediately after isolation and at fixed culture times, cells were incubated with 0.1 mM doxorubicin in Hanks' balanced salt solution for 10 min at 37 degrees C in 5% CO2-humidified atmosphere and observed by fluorescence microscopy. Parallel electron microscopy was performed to compare fluorescence data with general cell morphology. To monitor lysosomal acidification capacity, the fluorescent pH-sensitive vital dye LysoSensor-Blue was used. Doxorubicin fluorescence showed different patterns of nuclear and cytoplasmic staining, according to the time allowed for cell recovery and the culture method. In particular, cytoplasmic fluorescence changed from a diffuse staining, that could be observed after cell isolation and in hepatocyte suspensions, to a punctate perinuclear and pericanalicular fluorescence detectable in fully recovered hepatocyte monolayers. This study indicates that the 'doxorubicin-fluorescence test' may be considered a simple and rapid procedure for assessing hepatocyte functional condition. It may provide valuable and 'real time' guidelines for judging the correct way these cells are to be collected, preserved and utilized for clinical purposes.
    The Histochemical Journal 10/2000; 32(9):535-43.