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Published on: April 8, 2016
Isolation of lymphocytes from normal adult human liver suitable for phenotypic and functional characterization
M P Curry1, S Norris, L Golden-Mason
1Liver Unit, St. Vincent's University Hospital, Elm Park, Dublin, Ireland.
Insights
Optimizing liver lymphocyte isolation is crucial for studying their unique functions. A combined mechanical and enzymatic method preserves cell viability and function for flow cytometry and functional assays.
Area of Science:
- Immunology
- Hepatology
Background:
- Normal liver contains distinct resident lymphocytes.
- Characterizing these hepatic lymphocytes requires viable cells for analysis.
- Classical isolation methods may affect cell surface molecules and function.
Purpose of the Study:
- To evaluate the impact of isolation procedures on hepatic lymphocyte surface molecule expression and function.
- To optimize a technique for isolating viable hepatic lymphocytes with minimal alterations.
Main Methods:
- Mechanical homogenization and enzymatic digestion (collagenase type IV, DNase I) of human liver tissue.
- Flow cytometry for phenotypic characterization.
- In vitro functional assays including cytokine response, proliferation, and cytotoxicity.
Main Results:
- Combined mechanical and enzymatic dissociation improved viable lymphocyte yield compared to mechanical methods alone.
- A mean yield of 2.3 x 10^6 lymphocytes with 88.8% viability was achieved from human liver biopsies.
- Isolated lymphocytes were suitable for flow cytometry and retained in vitro growth, cytokine response, and cytotoxic functions.
Conclusions:
- A combined gentle mechanical dissociation followed by collagenase IV and DNase I digestion is an effective method for isolating viable hepatic lymphocytes.
- This optimized technique preserves lymphocyte phenotype and function for downstream analyses.
- The isolated cells are suitable for studying the distinct roles of liver-resident lymphocytes in immunity.
Abstract:
Murine and human studies have demonstrated that the normal liver contains significant numbers of resident lymphocytes that have functions distinct from those found in blood and other organs. To characterize these cells requires the isolation of viable lymphocytes that can be analysed by flow cytometry and in functional assays. The techniques classically used to isolate single cell suspensions of hepatic lymphocytes for phenotypic and functional studies involve mechanical and/or enzymatic dissociation of liver tissue. The aim of this study was to determine the effect of these procedures on surface molecule expression and lymphocyte function and to optimise an isolation technique that minimises these effects. Mechanical homogenisation of liver tissue alone resulted in low viable lymphocyte yields but these were improved by the combined use of mechanical and enzymatic techniques. A mean yield of 2.3 x 10(6) lymphocytes with a mean viability was 88.8% was obtained from 200 mg wedge biopsy samples of normal adult human liver using a combination of gentle mechanical dissociation followed by digestion with collagenase type IV and DNase I. These cells were suitable for phenotypic characterisation by flow cytometry. They also retained their ability to grow in vitro, to respond to cytokines and activation stimuli, to mediate cytotoxic killing of target cells, and to produce inflammatory and regulatory cytokines.
