Isolation of lymphocytes from normal adult human liver suitable for phenotypic and functional characterisation

Murine and human studies have demonstrated that the normal liver contains s ignificant: numbers of resident lymphocytes that have functions distinct fr om those found in blood and other organs. To characterise these cells requi res the isolation of viable lymphocytes that, can be analysed by how cytome try and in functional assays. The techniques classically used to isolate si ngle cell suspensions of hepatic lymphocytes for phenotypic and functional studies involve mechanical and/or enzymatic dissociation of liver tissue. T he aim of this study was to determine the effect of these procedures on sur face molecule expression and lymphocyte function and to optimise an isolati on technique that minimises these effects. Mechanical homogenisation of liv er tissue alone resulted in low viable lymphocyte yields but these were imp roved by the combined use of mechanical and enzymatic techniques. A mean yi eld of 2.3x10(6) lymphocytes with a mean viability was 88.8% was obtained f rom 200 mg wedge biopsy samples of normal adult human liver using a combina tion of gentle mechanical dissociation followed by digestion with collagena se type IV and DNase I. These cells were suitable for phenotypic characteri sation by flow cytometry. They also retained their ability to grow in vitro , to respond to cytokines and activation stimuli, to mediate cytotoxic kill ing of target cells, and to produce inflammatory and regulatory cytokines. (C) 2000 Elsevier Science B.V. All rights reserved.