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

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

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