P0105 : Changes in the CNS connexin 43 expression and function in the pathogenesis of hepatic encephalopathy

Abstract

should also target sinusoidal cells. However, there are no in vitro research tools that correctly mimic the unique features of the sinusoid: adequate spatial distribution of cells, biomechanical stimulation of the endothelial lineage and free paracrine interactions. Our aim was to design, fabricate and validate a three-dimensional co-culture chamber with microfluidics that mimics the hepatic sinusoid. Methods: A transparent PMMA chamber composed by 3 growing areas of 9.7 cm arranged at different heights and separated by 0.5mm was manufactured. The higher level integrated a microfluidic system allowing the application of homogeneous shear stress on a reinforced porous membrane where the endothelium is grown. The middle level had a second culture membrane, and on the lower level, the growing area was the base of the camera. Functional validation: Endothelial cells stimulated with shear stress (5 dyn/cm), HSC, and hepatocytes were cultured for 24h in the chamber. Morphology, viability and endothelial nitric oxide production was analyzed. Translational Validation: The applicability of the chamber in the field of cirrhosis was assessed co-culturing activated HSC and capillarized LSEC under shear stress, or in static conditions. Furthermore, the effects of adding the vasoprotective agent simvastatin were analyzed and compared with traditional culture methods. Results: Functional validation: Cells grown in the chamber showed excellent viability. Endothelial cells were aligned in the direction of shear stress, and increased their production of nitric oxide. Translational Validation: Dysfunctional LSEC cultured in the device showed a marked improvement in their phenotype (activation of the KLF2 pathway and decrease in endothelin-1) that was not observed using ordinary culture methods. The shear stress-derived improvement in LSEC led to a beneficial paracrine effect on HSC (reduced collagen I and alpha-SMA). Simvastatin addition produced a strong protective effect on both cell types, which was significantly higher than that obtained using traditional “transwell” methods. Conclusions: We herein describe a novel, versatile, easy to operate and highly reproducible device that can be applied in different fields of vascular biomedical research, including hepatology.