Purification and use of hepatocyte couplets.

Partial digestion of liver with collagenase can provide a cell population composed of single cells (singlets), pairs of cells (couplets) or larger groupings (multiplets) [l]. The individual cells within the couplets and multiplets possess both gap junction and tight junction function. Studies with these polarised primary cells can be aimed, for example, at the biliary pole without a requirement for long-term culture [1,2,3]. Because of presence of the large population (70%) of contaminating singlet cells, studies with hepatocyte couplets have been largely limited to microscopy. Examples are investigations of the distribution of fluorescent-labelled material [2,3,4] or electrophysiological investigations IS]. The present study was aimed at separating and purifying viable couplet (and triplet) cells from a mixed population of rat hepatocytes and is based on a centrifugal elutriation technique. This then offers an improved opportunity for biochemical studies of hepatobiliary function and malfunction. Hepatocyte units (singlets, couplets and multiples) were isolated from male Wistar rat liver by limited collagenase perfusion in situ based on the method described by Gautam & A. [l]. The method was modified by reducing the collagenase (c1. hvstolvticum, 0.287 U/mg, Boehringer Mannheim, Lewes) to 0.03% (w/v) and restricting the perfusion time to 4.5 min. The yield of couplet units was enhanced by further incubation of the remaining liver tissue at 37'C for 7 min. with 0.03% (w/v) collagenase solution. Cell viability was measured by the exclusion of trypan blue [6] and units were counted using an improved Neubauer haemocytometer. If a single cell within a unit was not viable, units were classified as non-viable even if one component cell was able to exclude trypan blue.