Bioartificial liver support has been focused on the therapy of severe liver failures. There are two problems in its development; 1) thrombus forms when blood contacts conventional polymer substrates and 2) hepatocytes lose hepatic functions rapidly in vitro. It is well-known that hepatic functions are maintained at high level when hepatocytes formed round shape. We have previously reported that poly(2-methoxyethyl acrylate) (PMEA) and poly(tetrahydrofurfuryl acrylate) (PTHFA) were blood compatible, although normal tissue cells can adhere on these polymers. Moreover, adhered cells exhibited round and spreading shapes on PMEA and PTHFA, respectively. It is expected that these blood-compatible polymers are able to control liver functions via the regulation of cell shape. Here, we investigated the adhesion and shapes of human hepatocyte model, HepG2, on PMEA and PTHFA to assess the possibility to apply these polymers as the substrates for bioartificial liver support.
Polymer-coated substrates were prepared by spin coating on polyethylene terephthalate (PET) disks. Adherent HepG2 were counted under an optical microscope after crystal violet staining. Focal adhesions were visualized after 1 day by immunofluorescent staining. The expression levels of hepatic genes, HNF4A and ALB, were measured by real time PCR analyses.
HepG2 adhered on PMEA and PTHFA as well as PET, indicating that hepatocytes can adhere on PMEA and PTHFA, although these polymers are blood compatible. To compare the shape of adhered HepG2, projected cell areas were measured after 1 day of culture. The projected cell areas were in the order of PTHFA > PET > PMEA. This result indicated that HepG2 kept round on PMEA, while the cells spread on PTHFA and PET. Additionally, HepG2 on PMEA expressed the highest hepatic genes among the polymers examined. These results indicated that hepatocytes on PMEA kept round shape and expressed high hepatic functions.
To assess the reason why HepG2 exhibited round shape and expressed high hepatic genes, we focused on integrin signaling and Hippo signaling pathways. Generally, the cells adhered on polymeric substrates via the interaction between integrin and adsorbed proteins. Surprisingly, HepG2 adhered on PMEA via both integrin-dependent and –independent manners and integrin signaling was weakened compared with the cells on PTHFA and PET. Integrin signaling promotes cell spreading. It seemed that weakened integrin signaling suppressed HepG2 spreading on PMEA. Moreover, YAP was located in nucleus on PTHFA but not PMEA. It has been reported that nuclear localization of YAP inhibits the expression of hepatic genes. Therefore, it seemed that nuclear localization of YAP on PTHFA inhibited the expression of hepatic genes.
Conclusively, PMEA is expected to be a new biomaterial to achieve the development of bioartificial liver support.