Cytotechnology 17: 127-131, 1995. 127 (~) 1995 KluwerAcademic Publishers. Printedin the Netherlands. Separation of intact and damaged hepatocytes in sucrose following non-enzymatic liver perfusion Alexander Yu. Petrenko, Alexander N. Sukach, Victor R Grischuk, Svetlana R Mazur and Andrey D. Roslyakov Institute for Problems of Cryobiology and Cryomedicine of the Ukrainian Academy of Sciences, 23 P ereyaslavskaya Street, Khrakov 310015 Ukraine Received6 June 1994;accepted3 April 1995 Key words: hepatocytes, isolation, membrane potential, respiratory activity, separation, xenobiotic metabolism Abstract This study deals with isolation of rat hepatocytes by a non-enzymatic method and the separation of intact and damaged cells in sucrose medium. Low speed centrifugation in isotonic sucrose medium of a hepatocyte suspension obtained by mechanical desaggregation of liver pre-perfused with EDTA solution results in the formation of a cell pellet which contains two different layers. A darker lower layer contains hepatocytes with intact plasma membranes. Their respiratory activity and xenobiotic metabolism are close to those of the cells isolated by collagenase perfusion. The study of distribution of lipophilic cation tetraphenylphosphonium (TPP +) indicates a predominantly mitochondrial localization of TPP + in the intact cells following non-enzymatic and collagenase isolation. Hepatocytes in the upper layer have damaged plasma membranes. As a result they lose the potential to accumulate TPP +, and have low rates of endogenous respiration and biotransformation activity. Addition of exogenous NADPH restores the capability to metabolize xenobiotics. Washing and incubation of these hepaticytes in an intracellular type medium results in restoration of uncoupler-stimulated oxygen consumption and generation of membrane potential in the presence of a succinate substrate. These properties are close to those of hepatocytes permeabilized by digitonin treatment. Thus, the procedure allows the simultaneous isolation of both intact and permeabilized hepatocytes with functionally active intracellular structures without the use of relatively expensive chemicals such as collagenase and Percoll. Abbreviations: 4-OHBP - 4-hydroxybiphenyl; BP - biphenyl; BSA - bovine serum albumin, DNP - 2,4- dinitrophenol; EDTA - ethylendiamintetraacetate; NADPH- nicotinamide adenine dinucleotide phosphate reduced; p-NA - p-nitroanisole; p-NPh - p-nitrophenol; TPP + - tetraphenylphosphonium. Introduction Though the enzymatic method is a routine technique for hepatocytes isolation (Berry, 1968; Seglen, 1976), successful attempts at non-enzymatic isolation of liver cells are known. It was reported (Berry et al., 1983) that perfusion of liver by solutions containing EDTA in the absence of collagenase with subsequent mechanical treatment resulted in the dissociation of liver into sin- gle cells, some of which had intact plasma membrane. After separation of the primary heterogeneous suspen- sion in Percoll intact hepatocytes were characterized by the level of ATR respiratory activity, metabolism of carbohydrates and nitrogen in comparison to cells isolated with collagenase (Berry et al., 1983). Dur- ing culture they synthesized albumin and triglycerides (Wang, 1985). The majority of cells in primary sus- pension had damaged plasma membranes and were discarded without investigation.