ECM-mimicking thermoresponsive surface for manipulating hepatocyte sheets with maintenance of hepatic functions

Hepatic cellular sheet-based tissue engineering is an attractive method for the treatment of liver diseases. However, cultured hepatocytes rapidly lose their viability and phenotypic functions on isolation from the native in vivo microenvironment of the liver. For providing extracellular matrix (ECM)-mimicking microenvironment to the isolated hepatocytes, poly(N-isopropylacrylamide) (PIPAAm)-grafted cell culture surface was functionalized with heparin, which has a similar structure to heparan sulfate on proteoglycans and possesses an affinity interaction with heparin-binding proteins such as heparin-binding EGF-like growth factor (HB-EGF). A cell cultivation system using HB-EGF bound heparin-immobilized thermoresponsive cell culture surface maintains the survival and adhesion of hepatocytes with highly hepatic functions such as albumin secretion. Bound HB-EGF stimulated EGF receptor and MAPK family ERK1/2 in dose-dependent manner. Time-course of HB-EGF stimulation revealed that the internalization of EGF receptors on heparin-immobilized surface was partially suppressed during a few days incubation. Sustained stimulation and activation of cultured hepatocytes on the HB-EGF bound surfaces were also confirmed. In addition, both the cultured hepatocytes and the growth factors detached as a contiguous cell sheet from the surface, accompanied by swelling and expanding of grafted thermoresponsive polymer. In conclusion, ECM-mimicking thermoresponsive cell culture surfaces facilitated the manipulation of hepatic cell sheets with maintaining hepatic functions by changing temperature. Creation of transferable and functional liver tissues is considered to have a potential to treat liver disease.