A practice-changing culture method relying on shaking substantially increases mitochondrial energy metabolism and functionality of human liver cell lines

<div><p>Practice-changing culturing techniques of hepatocytes are highly required to increase their differentiation. Previously, we found that human liver cell lines HepaRG and C3A acquire higher functionality and increased mitochondrial biogenesis when cultured in the AMC-Bioartificial liver (BAL). Dynamic medium flow (DMF) is one of the major contributors to this stimulatory effect. Recently, we found that DMF-culturing by shaking of HepaRG monolayers resulted in higher mitochondrial biogenesis. Here we further investigated the effect of DMF-culturing on energy metabolism and hepatic functionality of HepaRG and C3A monolayers. HepaRG and C3A DMF-monolayers were incubated with orbital shaking at 60 rpm during the differentiation phase, while control monolayers were maintained statically. Subsequently, energy metabolism and hepatic functionality were compared between static and DMF-cultures. DMF-culturing of HepaRG cells substantially increased hepatic differentiation; transcript levels of hepatic structural genes and hepatic transcription regulators were increased up to 15-fold (Cytochrome P450 3A4) and nuclear translocation of hepatic transcription factor CEBPα was stimulated. Accordingly, hepatic functions were positively affected, including ammonia elimination, urea production, bile acid production, and CYP3A4 activity. DMF-culturing shifted energy metabolism from aerobic glycolysis towards oxidative phosphorylation, as indicated by a decline in lactate production and glucose consumption, and an increase in oxygen consumption. Similarly, DMF-culturing increased mitochondrial energy metabolism and hepatic functionality of C3A cells. In conclusion, simple shaking of monolayer cultures substantially improves mitochondrial energy metabolism and hepatic differentiation of human liver cell lines. This practice-changing culture method may prove to prolong the <i>in-vitro</i> maintenance of primary hepatocytes and increase hepatic differentiation of stem cells.</p></div

DMF increases detoxification properties of HepaRG cells.

<p><b>(A)</b> Transcript levels of detoxification genes, n = 6-9/group. <b>(B-C)</b> CYP3A4 baseline- and induced-activity, n = 6-15/group. <b>(D)</b> Bile acid production, n = 6-15/group. <b>(E)</b> Transcript levels of hepatic transporter genes, n = 6-9/group. (F) TC uptake, n = 6/group. Significance Static <i>vs</i> DMF: # = <i>P</i> value <0.05, ## = <i>P</i> value <0.01, ### = <i>P</i> value <0.001 and #### = <i>P</i> value <0.0001.</p

DMF-culturing does not improve the polarization of the HepaRG cells.

<p><b>(A-B)</b> Staining of hepatic transporters OATP1a1(green) and MRP2 (red) with DAPI counter-staining for the nuclei (blue). <b>(C)</b> Visualization of CFDA (green) with DAPI counter-staining of the nuclei (blue), scale bar = 50μm.</p

DMF-culturing enhances the differentiation of HepaRG cells.

<p><b>(A)</b> Staining of albumin (green), and SOX9 (red), with DAPI counter-staining of the nuclei (blue) in HepaRG-Static and HepaRG-DMF. The arrow indicates the nuclear translocation of SOX9 in HepaRG-Static, scale bar = 50μm. <b>(B)</b> Albumin synthesis. <b>(C)</b> Transcript levels of genes encoding hepatic transcription regulators, n = 6-9/group. Significance Static <i>vs</i> DMF: # = <i>P</i> value <0.05, ## = <i>P</i> value <0.01, ### = <i>P</i> value <0.001 and #### = <i>P</i> value <0.0001. <b>(D)</b> Staining for CEBPα (red), with DAPI counter-staining of the nuclei (blue). The arrow indicates nuclear translocation of CEBPα, observed in HepaRG-DMF, scale bar = 50μm.</p

DMF-culturing enhances mitochondrial energy metabolism and hepatic functions of C3A cells.

<p><b>(A-B)</b> Lactate production and glucose consumption, n = 6/group. <b>(C)</b> Transcript levels of genes encoding hepatic differentiation regulators, n = 6/group. (D) Albumin synthesis. <b>(E-F)</b> CYP3A4 activity, n = 3/group. <b>(G)</b> Transcript levels of genes involved in nitrogen metabolism, n = 6/group. <b>(H-I)</b> Ammonia elimination and urea production, n = 3-6/group. Significance Static <i>vs</i> DMF: # = <i>P</i> value <0.05, ## = <i>P</i> value <0.01, ### = <i>P</i> value <0.001 and #### = <i>P</i> value <0.0001.</p

DMF increases mitochondrial energy metabolism of HepaRG cells.

<p><b>(A)</b> Transcript levels of <i>PGC1a</i> and <i>MT-CYB</i>, n = 6/group. <b>(B-D)</b> Lactate production, glucose and oxygen consumption, n = 9-15/group. Significance Static <i>vs</i> DMF: # = <i>P</i> value <0.05, ## = <i>P</i> value <0.01, ### = <i>P</i> value <0.001 and #### = <i>P</i> value <0.0001.</p

Efficacy of HepaRG-AMC-BAL treatment of rats with ALF.

<p>Efficacy was demonstrated by a significantly increased survival time (<i>P</i> = 0.001) (A), the median clinical grading score for HE (B), increased time to reach clinical HE score 4 (<i>P</i> = 0.005) (C), lower blood ammonia levels (D), and lower plasma creatinine levels (E). Continuous lines indicate the control group and dotted lines indicate the experimental group. Values are expressed as median scores (B), or means ± standard deviations (D, E) (n = 5 to 6). Significance: * <i>P</i><0.05 <i>versus</i> control group.</p

Clinical grading score of hepatic encephalopathy.

<p>Clinical grading score of hepatic encephalopathy.</p

Transcript levels of −DMSO and +DMSO HepaRG-AMC-BALs.

<p>Transcript levels are indicated as % of mean mRNA levels of two human liver samples and normalized for 18S ribosomal RNA. The change in transcript levels of +DMSO BALs relative to the −DMSO BALs are indicated with ↑ for upregulation and ↓ for downregulation. Abbreviations: DMSO, dimethyl sulfoxide; AMC-BAL, Academic Medical Center-bioartificial liver. Values are given as means ± standard deviations (n = 5 to 6). <i>P</i> values refer to −DMSO <i>versus</i> +DMSO BALs.</p

HA stainings of cross sections of −DMSO (A, C) and +DMSO HepaRG-AMC-BALs (B, D).

<p>Full transverse sections (A, B) show the spirally wound matrix layers with the gas capillaries (arrows) positioned in between. Details of the matrix (C, D) show the polyester matrix fibers (arrowheads) with HepaRG cells, and the web-shaped extracellular matrix in acellular areas in the matrix (D). Bars: 200 µm (A, B) and 20 µm (C, D).</p