# Protocol for optimized mononuclear cell isolation from liver and tumor tissue using mechanical or enzymatic digestion

## TL;DR

This paper provides a detailed protocol for isolating immune cells from liver and tumor tissue using mechanical or enzymatic methods.

## Contribution

The study introduces an optimized protocol for isolating mononuclear cells from liver tissue with reduced experimenter bias.

## Key findings

- The protocol enables efficient digestion and isolation of immune cells from both tumor and non-tumor liver regions.
- Both mechanical and enzymatic digestion techniques are evaluated using qualitative and quantitative measurements.

## Abstract

Mononuclear cells from human liver tissue, including tumor and non-tumor regions, are a valuable source for studies of the tumor microenvironment. Here, we present a protocol to dissociate liver tissue from patients with hepatocellular carcinoma. We provide steps to generate single-cell suspension using either mechanical or enzymatic digestion, following steps for separating mononuclear cells using density gradient. Finally, we describe steps for flow cytometry staining of isolated immune cells.

For complete details on the use and execution of this protocol, please refer to Moreno-Fernandez et al.1 and Heinrich et al.2

•Steps from resected tissue to single-cell suspension of immune cells•Instructions for efficient digestion and isolation with reduced experimenter bias•Evaluation of both techniques using qualitative and quantitative measurements•Guidance on analyses of low-abundance immune cells

Steps from resected tissue to single-cell suspension of immune cells

Instructions for efficient digestion and isolation with reduced experimenter bias

Evaluation of both techniques using qualitative and quantitative measurements

Guidance on analyses of low-abundance immune cells

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Mononuclear cells from human liver tissue, including tumor and non-tumor regions, are a valuable source for studies of the tumor microenvironment. Here, we present a protocol to dissociate liver tissue from patients with hepatocellular carcinoma. We provide steps to generate single-cell suspension using either mechanical or enzymatic digestion, following steps for separating mononuclear cells using density gradient. Finally, we describe steps for flow cytometry staining of isolated immune cells.