LPS induces TNF-α production in PMA-treated THP-1 cells.

<p>THP-1 were treated with 0, 5, 10 and 25 ng/mL PMA for 48h prior exposure to 1 μg/mL LPS. The concentration of TNF-α was measured from the 48h culture supernatants by ELISA. These findings indicate that PMA differentiated THP-1 cells are well differentiated and yet respond adequately to a subsequent low-concentration of LPS. Values are presented as duplicates.</p

Gene expression of fibrotic markers and cytokines in human liver microtissues exposed to LPS, TNF-α and TGF-β1.

<p>mRNA was extracted using TRIzol conventional procedure and fold induction were calculated as 2^(-ΔΔCT) for each sample and vehicle control and expressed as mean fold induction ± S.E.M of three replicates with six MTs each. Actin was used as reference gene for each sample. † pool of 16 microtissues analysed as duplicate; no statistical analysis were performed on these samples. ND: no-detected values. *; P ≤ 0.05, **; P ≤ 0.01, ***; P ≤ 0.001 vs vehicle control.</p

Immunostaining of formalin fixed paraffin embedded human microtissues after exposure to MTX, TAA and TGF-β1.

<p>Formalin fixed paraffin embedded slides of HepaRG/THP-1 macrophages/hTERT-HSC microtissues were stained with Hematoxylin & Eosin (H&E), vimentin, α-smooth muscle actin (αSMA), collagen I and CD68 after 14 days of treatment with MTX, TAA and TGF-β1. Microtissues were fixed in 4% PFA and embedded in 2% agarose prior paraffinization. Microtissues showed increase in the vimentin, αSMA, collagen I and CD68 positive cells after MTX, TAA and TGF-β1 exposure. Vimentin and CD68 stainings show proliferation of stellate cells and THP-1 macrophages in the microtissues, suggesting the onset of inflammation process, while αSMA and collagen I indicate activation of stellate cells and deposition of collagen. Scale bar: 200μm. Graphics show Quantitative IHC Staining Value (QISV) as mean ± S.D. (N = 5). *; P ≤ 0.05, **; P ≤ 0.01, ***; P ≤ 0.001 vs vehicle control.</p

Albumin production and cell proliferation in liver microtissues.

<p>Formalin fixed paraffin embedded slides of HepaRG/THP-1 macrophages/hTERT-HSC microtissues were stained with Albumin and Ki67 antibodies after 14 days of treatment with MTX, TAA and TGF-β1. Microtissues were fixed in 4% PFA and embedded in 2% agarose prior paraffinization. Microtissues showed decrease in albumin production after MTX, TAA and especially TGF-β1 exposure. Ki67 shows strong induction of cell proliferation in the microtissues after TGF-β1 exposure. Scale bar: 200μm. Graphics show Quantitative IHC Staining Value (QISV) as mean ± S.D. (N = 5). *; P ≤ 0.05, **; P ≤ 0.01, ***; P ≤ 0.001 vs vehicle control.</p

TNF-α and TGF-β1 promote αSMA production in monolayer culture of hTERT-HSC.

<p>hTERT-HSC cells were treated for 2, 5 or 10 days with TNF-α (50ng/mL) or TGF-β1 (1ng/mL). After treatment, the cells were fixed and stained against αSMA (green) and nuclei (DAPI, blue). Pictures taken using fluorescence microscopy. Scale bar: 50μm.</p

Viability response of human liver microtissues treated with pro-fibrotic compounds.

<p>(A) Effect of LPS, TNF-α and TGF-β1 on viability of human liver microtissues was assessed by MTT assay. Microtissues were incubated with 0.5mg/mL MTT solution for 4h after 14 days of exposure to the tested compounds. DMSO and Sörensen buffer were then added into the wells and absorbance was read at 550nm using FlexStation 3. Values are expressed as percentage of negative control. *; P ≤ 0.05, **; P ≤ 0.01, ***; P ≤ 0.001 vs vehicle control. (n = 6, mean ± SD). (B) Effect of MTX, TAA and TGF-β1 on the ATP production. ATP content was measured using CellTiter-Glo^® Luminescent Cell Viability Assay 2.0 after 14 days of exposure to MTX, TAA and TGF-β1. Values are expressed as percentage of negative control. ***; P ≤ 0.001 vs vehicle control (n = 6, mean ± SD).</p

Staining of formalin fixed paraffin embedded human microtissues generated with HepaRG-cells or HepaRGs/THP-1/hTERT-HSC.

<p>Microtissues were stained with Hematoxylin & Eosin (H&E), mesenchymal NPC-marker vimentin and macrophages marker CD68. Microtissues were kept in culture during 14 days before performing histological staining. Co-culture systems showed positive staining for vimentin and CD68 indicating the presence of the three different cell types. Arrows indicate CD68-positive cells. Dendritic stellate cells are shown in the zoom of vimentin staining picture. Scale bar: 200μm (20X magnification) and 100μm (40X magnification).</p

Secretion of Collagen I and MMP2 in supernatant medium after exposure of microtissues to pro-fibrotic compounds.

<p>Protein amount was assessed by ELISA in supernatant medium after 14 days of exposure to MTX, TAA and TGF-β1. *; P ≤ 0.05, **; P ≤ 0.01, ***; P ≤ 0.001 vs vehicle control (n = 4, mean ± SD).</p

Effect of MTX and TAA on gene expression of fibrotic markers in human liver microtissues.

<p>mRNA was extracted using TRIzol conventional procedure and fold induction were calculated as 2^(-ΔΔCT) for each sample and negative control and expressed as mean fold induction ± S.E.M. of three replicates with six MTs each. Actin was used as reference gene for each sample. *; P ≤ 0.05, **; P ≤ 0.01, ***; P ≤ 0.001 vs vehicle control.</p

TaqMan probes used for the research.

<p>TaqMan probes used for the research.</p