All-In-One: Advanced preparation of Human Parenchymal and Non-Parenchymal Liver Cells.

BACKGROUND & AIMS: Liver cells are key players in innate immunity. Thus, studying primary isolated liver cells is necessary for determining their role in liver physiology and pathophysiology. In particular, the quantity and quality of isolated cells are crucial to their function. Our aim was to isolate a large quantity of high-quality human parenchymal and non-parenchymal cells from a single liver specimen. METHODS: Hepatocytes, Kupffer cells, liver sinusoidal endothelial cells, and stellate cells were isolated from liver tissues by collagenase perfusion in combination with low-speed centrifugation, density gradient centrifugation, and magnetic-activated cell sorting. The purity and functionality of cultured cell populations were controlled by determining their morphology, discriminative cell marker expression, and functional activity. RESULTS: Cell preparation yielded the following cell counts per gram of liver tissue: 2.0 ± 0.4 × 10(7) hepatocytes, 1.8 ± 0.5 × 10(6 )Kupffer cells, 4.3 ± 1.9 × 10(5) liver sinusoidal endothelial cells, and 3.2 ± 0.5 × 10(5) stellate cells. Hepatocytes were identified by albumin (95.5 ± 1.7%) and exhibited time-dependent activity of cytochrome P450 enzymes. Kupffer cells expressed CD68 (94.5 ± 1.2%) and exhibited phagocytic activity, as determined with 1 µm latex beads. Endothelial cells were CD146(+) (97.8 ± 1.1%) and exhibited efficient uptake of acetylated low-density lipoprotein. Hepatic stellate cells were identified by the expression of α-smooth muscle actin (97.1 ± 1.5%). These cells further exhibited retinol (vitamin A)-mediated autofluorescence. CONCLUSIONS: Our isolation procedure for primary parenchymal and non-parenchymal liver cells resulted in cell populations of high purity and quality, with retained physiological functionality in vitro. Thus, this system may provide a valuable tool for determining liver function and disease.

Chemical modifications on siRNAs avoid Toll-like-receptor-mediated activation of the hepatic immune system in vivo and in vitro.

OBJECTIVES: The therapeutic application of small interfering RNAs (siRNAs) is limited by the induction of severe off-target effects, especially in the liver. Therefore, we assessed the potential of differently modified siRNAs to induce the hepatic innate immune system in vitro and in vivo. METHODS: Primary isolated liver cells were transfected with siRNAs against apolipoprotein B1 (APOB1), luciferase (LUC) or galactosidase (GAL). For in vivo use, siRNAs were formulated in lipid nanoparticles (LNPs) and administered intravenously to C57BL/6 mice. Liver tissue was collected 6-48 h after injection and knock-down efficiency or immune responses were determined by quantitative reverse-transcription-linked PCR. RESULTS: Unmodified GAL siRNA transiently induced the expression of TNF-α, IL-6, IL-10, IFN-ß and IFN-sensitive gene 15 in vivo, whereas a formulation of 2'-O-methylated-LUC siRNA had no such effects. Formulation of unmodified APOB1-specific siRNA suppressed APOB1 mRNA levels by ~80% in the liver 48h after application. The results were paralleled in vitro, where transfection of liver cells with unmodified siRNAs, but not with chemically modified siRNAs, led to cell-type-specific induction of immune genes. These immune responses were not observed in MYD88-deficient mice or in chloroquine-treated cells in vitro. CONCLUSIONS: Our data indicate that siRNAs activate endosomal Toll-like receptors in different liver-derived cell types to various degrees, in vitro. LNP-formulated siRNA selectively leads to hepatic knock-down of target genes in vivo. Here, off-target immune responses are restricted to non-parenchymal liver cells. However, 2'-O-methyl modifications of siRNA largely avoid immune-stimulatory effects, which is a crucial prerequisite for the development of safe and efficient RNA-interference-based therapeutics.

Corticosteroids shift the Toll-like receptor response pattern of primary-isolated murine liver cells from an inflammatory to an anti-inflammatory state.

OBJECTIVE: Only little is known about the mechanisms of action of corticosteroids in the treatment of inflammatory liver diseases. As there is increasing evidence that stimulation of the innate immune system plays an important pathogenetic role in these conditions, we hypothesized that steroids may interfere with the activation of the Toll-like receptor (TLR) system of the liver. METHODS: To test this hypothesis, murine non-parenchymal liver cells (Kupffer cells, liver sinusoidal endothelial cells) and primary hepatocytes were stimulated with TLR 1-9 ligands in the presence or absence of dexamethasone. Expression of pro- and anti-inflammatory cytokines was determined by quantitative reverse transcription-PCR or ELISA, respectively. Nuclear factor 'kappa-light-chain-enhancer' of activated B-cells (NF-κB) activation was assessed by western blot analysis. RESULTS: TLR agonists induced the expression of pro- [tumor necrosis factor-α (TNF-α), IL-6, IL-1ß, IFN-ß] and anti-inflammatory cytokines [IL-10, transforming growth factor-ß (TGF-ß)], which was differentially modulated by steroid treatment. TNF-α and IL-6 expression was suppressed by dexamethasone, while IL-10 but not TGF-ß was enhanced after TLR stimulation. IFN-ß production induced by TLR 4 agonists but not TLR 3 agonists was inhibited by dexamethasone. TLR expression itself was down-regulated by steroid treatment in a cell type-specific manner. These effects were associated with suppression of the TLR-mediated activation of NF-κB. CONCLUSIONS: TLR signaling is modulated by corticosteroids in a cell type-specific fashion resulting in down-regulation of TLR expression, suppression of pro-inflammatory and up-regulation of anti-inflammatory cytokines. This represents an as yet unknown mechanism of action for corticosteroids that may at least in part explain their therapeutic effects in inflammatory liver diseases.