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+/-0.4x107 hepatocytes, 1.8+/-0.5x106 Kupffer cells, 4.3+/-1.9x105 liver sinusoidal endothelial cells, and 3.2+/-0.5x105 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 1mum 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 alpha-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

Quantitative analysis of the purities of cultured cell populations.

<p>Abbreviations: Primary human hepatocytes (PHH); Kupffer cells (KC); Liver sinusoidal endothelial cells (LSEC); Hepatic stellate cells (HSC); Cluster of differentiation (CD); α-smooth muscle actin (α-SMA); Standard error of mean (SEM).</p><p>Quantitative analysis of the purities of cultured cell populations.</p

Functional activity of cultured cells.

<p>In PHH activity of CYP3A4 was determined after stimulation with 25μM dexamethasone for 6-48h. In addition CYP3A4 was inhibited by treatment with 2% DMSO. Cells were treated with ETOH or DMSO as negative controls. Relative light units (RLU) are given as mean±SEM (n = 3). Furthermore, CYP3A4 and albumin fluorescence imaging revealed a heterogeneous pattern of cultured PHH (A). KC exhibited strong phagocytic activity by the uptake of 1μm fluorescent latex beads (green) in a time-dependent manner (B). LSEC exhibited efficient endocytic capability, shown by the efficient incorporation of AcLDL (green) after 1h of incubation which was metabolized after incubation for 6h (C). HSC, cultured for a short time, were visualized by retinol (vitamin A) autofluorescence signals. Brightness reinforcement (BrightR) detection mode was used to amplify dim structures and make them accessible. To distinguish HSC from myofibroblasts, CYGB (green) and α-SMA (red) were fluorescently stained in the HSC population. Nuclei were stained with DAPI (blue). Images were taken at 40× magnification. Scale bar, 50μm. Furthermore, RNA was extracted from freshly isolated (uncultured) HSC and HSC cultured for 10 days (n = 6). Gene expression of <i>ACTA2</i>, <i>COL1A1</i> and <i>LOXL2</i> was determined by RT-qPCR. Data represent mean of copy numbers (mean±SEM) normalized to the reference gene <i>ACTB</i> (D). Asterisks indicate significant results (* p<0.05; ** p<0.01; *** p<0.001).</p

Identification of PHH and NPC.

<p>Cell morphology was examined by phase contrast microscopy using an EVOS^TM XL Core Imaging System (AMG). Isolated cell populations were identified by immunofluorescence staining of cell type—specific markers (n = 5). Nuclei were counterstained with DAPI (blue). Scale bar, 50μm. In addition, RNA was extracted from liver cells (n = 10) and gene expression of cell type-specific markers was determined by RT-qPCR. Copy numbers were normalized to the reference gene <i>ACTB</i>. The expression of cell markers in the reference cell type was defined as 100% (e.g. APOB in PHH). The expression of these markers in the other cell types is expressed as fold change in %. PHH exhibited a binucleated, polygonal shape (A) and strongly expressed albumin (B, red). In addition, PHH showed <i>APOB</i> and <i>ASGR1</i> gene expression (C). KC exhibited an irregular morphology (D) and expressed CD68 on protein level (E, red). Furthermore KC showed <i>CD163</i> and <i>CD14</i> gene expression (F). LSEC formed their unique morphology (G) and were identified by staining of CD146 (H, red). LSEC expressed the markers <i>PECAM1</i>, <i>VWF</i> and <i>LYVE-1</i> (I). HSC changed into myofibroblast-like cells (J) and were identified with anti-α-SMA antibody (K, green). Moreover, HSC were characterized by the gene expression of the markers <i>VCL</i>, <i>DES</i> and <i>CYGB</i> (L).</p