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Updated: May 13, 2026
Protocol for Isolation of Primary Human Hepatocytes and Corresponding Major Populations of Non-parenchymal Liver Cells
Published on: March 30, 2016
NK cell isolation from liver biopsies: phenotypic and functional analysis of low cell numbers by flow cytometry
Ning Li1, Gisella L Puga Yung, Amandine Pradier
1Division of Clinical Immunology and Allergology, Department of Medical Specialties, University Hospital and Medical Faculty Geneva, Switzerland.
Insights
This study refines methods for isolating intrahepatic lymphocytes (IHL) from liver biopsies (LB), enabling reliable analysis of natural killer (NK) cells in liver disease research. The optimized protocol yields sufficient viable NK cells for phenotype and function assessment from small tissue samples.
Area of Science:
- Immunology
- Hepatology
- Cell Biology
Background:
- Natural killer (NK) cells are crucial in liver disease pathogenesis.
- Limited numbers of intrahepatic lymphocytes (IHL) from liver biopsies (LB) hinder ex vivo analysis.
- Existing isolation methods may compromise IHL yield, viability, phenotype, and function.
Purpose of the Study:
- To refine and evaluate a modified isolation protocol for cell yields and viability from needle LB.
- To assess the impact of mechanical dissociation and enzymatic digestion on IHL phenotype and function.
- To establish reliable methods for analyzing very low cell numbers of intrahepatic NK cells.
Main Methods:
- Developed and validated a modified isolation protocol for IHL from standard needle LB specimens (mean weight 9.1 mg).
- Utilized flow cytometry for phenotypic analysis of NK cells (CD56, NKp46, CD16).
- Assessed NK cell function via degranulation (CD107a) and cytokine production (IFN-γ) after in vitro stimulation, miniaturizing assays for low cell numbers.
Main Results:
- Obtained a mean of 7,364 IHL/mg with >90% viability from LB.
- Enzymatic digestion (collagenase IV, DNase I) did not affect IHL viability or NK cell function, though CD56 surface expression decreased.
- Reliable phenotypic characterization was achieved with as few as 2,500 IHL, and functional assays were miniaturized for 25,000 IHL.
Conclusions:
- The refined protocol enables reliable phenotypic and functional analysis of intrahepatic NK cells from small LB specimens.
- This method provides a valuable tool for studying human NK cell immunobiology in liver diseases.
- The findings facilitate a deeper understanding of NK cell roles in the liver microenvironment.
Abstract:
Natural killer (NK) cells are considered to play a critical role in liver disease. However, the available numbers of intrahepatic lymphocytes (IHL) derived from liver biopsies (LB) for ex vivo analysis of intrahepatic NK cells is very limited; and the isolation method may hamper not only yields and viability, but also phenotype and function of IHL. The aim of the present study was therefore to (1) refine and evaluate the cell yields and viability of a modified isolation protocol from standard size needle LB; and (2) to test the effects of mechanical dissociation and enzymatic tissue digestion, as well as the analysis of very low cell numbers, on the phenotype and function of intrahepatic NK cells. Peripheral blood mononuclear cells (PBMC) and IHL, freshly isolated from the peripheral blood, LB (n = 11) or partial liver resections (n = 5), were used for phenotypic analysis by flow cytometry. NK cell function, i.e., degranulation and cytokine production, was determined by staining of CD107a and intracellular IFN-γ following in vitro stimulation. The mean weight of the LB specimens was 9.1 mg, and a mean number of 7,364 IHL/mg were obtained with a viability of >90%. Exposure of IHL and PBMC to 0.5 mg/ml collagenase IV and 0.02 mg/ml DNase I for 30 min did affect neither the viability, NK cell function, nor the percentages of CD56(+), NKp46(+), and CD16(+) NK cells, whereas the level of CD56 surface expression was reduced. The phenotype of LB-derived NK cells was reliably characterized by acquiring as few as 2,500 IHL per tube for flow cytometry. The functional assay of intrahepatic NK cells was miniaturized by culturing as few as 25,000 IHL in 25 μl (10(6)/ml) using 96-well V-bottom plates with IL-2 and IL-12 overnight, followed by a 4 h stimulation with K562 cells at a NK:K562 ratio of 1:1. In summary, we report reliable phenotypic and functional analyses of small numbers of intrahepatic NK cells isolated from LB specimens providing us with a tool to better address the emerging role of human NK cell immunobiology in liver diseases.
