Protocol for isolating CD163+ Kupffer cells from human liver resections

Abstract

The liver microenvironment contains a wide variety of monocyte and macrophage populations. Here, we present a protocol for the specific isolation of liver-resident macrophages, known as Kupffer cells (KCs), from human liver resections. We describe steps for dissociating human liver tissues, separating non-parenchymal cells into fractions by a 2-phase iodixanol gradient, and positive selection of KCs based on the expression of CD163. We then provide instructions for validating the procedure by immunofluorescence to detect CD163. For complete details on the use and execution of this protocol, please refer to Roca Suarez, Plissonnier, et al.¹.

Keywords: Cell Biology; Cell isolation; Immunology.

Graphical abstract

Figure 1

Liver dissociation and isolation of CD163⁺ KCs by magnetic separation (A) Expression of CD163 protein in each cell population of the human liver microenvironment. Data obtained from Guilliams et al., Cell 2022. (B) KC isolation method based on a 2-phase iodixanol gradient and CD163⁺ selection. (C) Placement of tubes in selected vessels in order to perform washing and enzymatic dissociation of the liver tissue. (D) Mechanical dissociation and filtering of liver tissues. (E) Dissociated liver tissues following centrifugation, where the NPC fraction can be observed in the supernatant above the hepatocyte pellet. (F) 2-phase iodixanol gradient following centrifugation, showing a separation between HSCs (top layer) and the monocytes/macrophages + LSECs fraction (bottom layer). (G) Experimental setup for the magnetic selection of CD163⁺ KCs, showing a pre-separation filter (yellow) on top of a LS column that is attached to a MidiMACS separator. (H) Microscopic image of human KCs cultured for 24 h following isolation. HSCs, hepatic stellate cells; KCs, Kupffer cells; LSECs, liver sinusoidal endothelial cells; NPC, non-parenchymal cells; PHH, primary human hepatocytes.

Figure 2

Validation of the KC isolation procedure by IF (A) IF microscopy image of KCs, showing positive staining for nuclear DNA (DAPI, blue), CD68 (red) and CD163 (green). (B) Percentage of CD163⁺/CD68⁺ KCs among total cells obtained following the isolation procedure (three independent experiments, two-three fields each). Bars represent mean ± SD. Images obtained from Roca Suarez, Plissonnier et al., Gut 2024. IF, immunofluorescence; KCs, Kupffer cells.

Figure 3

Induction of an inflammatory response via TLR8 signaling in KCs (A) Treatment of KCs with the TLR8 agonist SLGN (150 nM, 24 h) induces the upregulation of monocyte genes and the downregulation of KC markers and TFs (n = 3, FDR<0.05, GEO: GSE240054). (B) Concentration of cytokines present in culture supernatants from KCs stimulated with SLGN or DMSO (n = 4, Mann-Whitney test). Bars represent mean ± SEM. Data obtained from Roca Suarez, Plissonnier et al., Gut 2024. AQP9, aquaporin 9; CCL, C-C motif chemokine ligand; CD5L, CD5 molecule like; DMSO, dimethyl sulfoxide; DNMT3A, DNA methyltransferase 3 alpha; EREG, epiregulin; FOLR2, folate receptor beta; ID3, inhibitor of DNA binding 3; IFN, interferon; IL, interleukin; KCs, Kupffer cells; S100A, S100 calcium binding protein A; SLC, solute carrier family; SLGN, selgantolimod; SPIC, Spi-C transcription factor; TFs, transcription factors; TFEC, transcription factor EC; TIMD4, T cell immunoglobulin and mucin domain containing 4; TNF-α, tumor necrosis factor alpha; TLR8, Toll-like receptor 8; VSIG4, V-set and immunoglobulin domain containing 4.