Protocol to study monocyte transmigration across primary human liver endothelial cells under physiological shear flow conditions in vitro

Abstract

Modeling immune cell recruitment by liver endothelial cells in vitro is important to better understand the pathology of chronic inflammatory liver diseases and cancers. Here, we present a protocol for the study of monocyte transmigration across activated primary human liver endothelial cells, under physiological flow conditions. We describe primary endothelial cell isolation from human liver tissues and monocyte isolation from human blood. We then detail the shear flow-based assay and subsequent analysis of the different stages of monocyte transmigration. For complete details on the use and execution of this protocol, please refer to Wilkinson et al.¹.

Keywords: Cell isolation; Cell-based Assays; Immunology.

Graphical abstract

Figure 1

Isolation of primary liver endothelial cells from fresh human liver tissue (a diseased sample is shown) (A) (i) Liver slices from donor or diseased explants are (ii) mechanically chopped and (iii) enzymatically digested and (iv) strained through a fine mesh. (i) White arrow indicates a vascular area rich in connective tissue. (B) The digest is concentrated through a series of centrifugation steps to remove debris. (C) The digest is layered on top of a 33:77% Percoll gradient and (ii) the non-parenchymal cell fraction (white arrow) is isolated by density centrifugation. (iii and iv) Non-parenchymal cells are washed with phosphate buffered saline by centrifugation. (D) Biliary epithelial cells (BEC) and immune cells are removed by immunomagnetic selection against (i) epithelial cell adhesion molecule (EpCAM) and (ii) CD45, respectively. (iii) Liver endothelial cells are then isolated by positive immunomagnetic selection for CD31. (E) Cells are placed in culture in the presence of vascular endothelial growth factor and hepatocyte growth factor and passaged up to a maximum of five passages (p5). Scale bar represents 400 μm.

Figure 2

Isolation of monocytes from fresh human blood (A) Whole blood is carefully layered on top of Lympholyte-H Cell Separation Media. (B) The buffy layer (white arrow), containing peripheral blood mononuclear cells (PBMCs), is collected, transferred to a new 15 mL and PBMCs are washed in MACS buffer and counted. (C) Non-monocytes are labeled with the antibody cocktail and magnetic beads included in the Pan Monocyte isolation kit (Human; Miltenyi) (D and E) Monocytes are negatively selected out of the PBMC suspension by passing the PBMC suspension through an LS Separation column in the magnetic field of a MidiMACS separator. (F) Purified monocytes are collected and counted before being used in the shear flow-based assay.

Figure 3

Evaluation of monocyte viability and purity Flow cytometry histograms of percentage live monocytes (left) and CD14⁺ monocytes (right). Red peak represents the isotype control.

Figure 4

Identification of transmigration stage of monocytes across primary human liver endothelial cells Primary human liver endothelial cells are stimulated for 24 h with the senescence-associated secretory phenotype (SASP) collected from cells undergoing oncogene-induced senescence (ER:HRas^G12V IMR90 cells; Ras) and growing cell control supernatants (Grow). (A) Identifying adhered (black square), shape-changed (red square) and transmigrated (dotted square) monocytes via their morphology and phase brightness/darkness. Scale bar represents 25 μm. (B) Mean counts per lane are normalized to cells/mm²/10⁶ cells perfused (upper panel) and shape-changed and transmigrated monocyte numbers are expressed as a percentage of total adherent cells (lower panel). Here, we show that primary human liver endothelial cells activated with Ras supernatant support significantly higher levels of monocyte adherence and transmigration, when compared to controls treated with Grow supernatant. ∗ and ∗∗ indicate statistical significance, where p ≤ 0.05 or p ≤ 0.01, respectively. ns = not significant.

Figure 5

Identification of monocyte transmigration route across primary human liver endothelial cells (A) Monocytes (blue; white arrows) adhered to and migrating across a monolayer of liver endothelial cells (green). Endothelial cell-cell junctions are delineated with immunofluorescent staining of VE-cadherin (red). Scale bar represents 50 μm. (B) Transmigrating monocyte (blue; white arrow) utilizing the paracellular pathway between liver endothelial cells (green) causes a visible break in the VE-cadherin (white). (C) Example of a lymphocyte (blue; white arrow) transmigrating across a liver endothelial cell monolayer (green) via the transcellular pathway, which is characterized by an enrichment of F-actin (red). Scale bar represents 10 μm.