Switching of growth factor binding on heparin-functionalized thermoresponsive surface for hepatocyte sheet manipulation with maintenance of hepatic functions

摘要

Introduction: Liver tissue engineering using primary hepatocytes is an attractive method for the treatment of liver diseases. Engineered Hepatocyte sheets were effectively engrafted in pre-vascularized subcutaneous site and exhibited liver-specific functionalities. By contrast, cultured hepatocytes rapidly lose their viability and phenotypic functions on isolation from the native in vivo microenvironment of the liver. To overcome this problem, heparin-functionalized poly(N-isopropylacrylamide) (PIPAAm)-grafted cell culture surface, which interacts with heparin-binding proteins such as heparin-binding EGF-like growth factor (HB-EGF), was been designed for maintaining hepatic functions during the cultivation (Figure 1, left). In addition, the detachment of the cultured hepatocytes as a sheet was examined when lowering temperature to 20°C (Figure 1, right). Figure 1. Schematic of affinity regulation between heparin-binding protein and immobilized heparin by conformational change of PIPAAm. Materials and Methods: Heparin-functionalized thermoresponsive surfaces were prepared as described previously. Briefly, poly(IPAAm-co-2-carboxyisopropylacrylamide) (poly(IPAAm-co-CIPAAm))-grafted surfaces on tissue culture polystyrene (TCPS) dishes were prepared by electron beam irradiation. Heparin was covalently immobilized on the poly(IPAAm-co-CIPAAm)-grafted dishes by condensing reaction. Then, affinity binding of HB-EGF was performed on the heparinized thermoresponsive surfaces by incubation of HB-EGF solution at 37°C for 24 h. Amounts of bound HB-EGF on the heparinized surface were quantified using [~(125)I]-labeled HB-EGF. Primary rat hepatocytes were seeded on the dishes in DMEM with 10% fetal bovine serum (FBS) at 37°C in a humidified atmosphere with 5% CO_2. Results: The addition of soluble HB-EGF in the cell culture media was essential for the survival of hepatocytes. When the medium contained less than 10 ng/cm~2 of soluble HB-EGF, the hepatocytes were not able to adhere and form their cell sheets. By contrast, hepatocytes adhered and formed their sheets on heparin-functionalized thermoresponsive surface with 10 ng/cm~2 of bound HB-EGF. In addition, the secretion of albumin on bound HB-EGF was maintained and higher than that on PIPAAm-grafted surfaces with soluble HB-EGF. Therefore, bound HB-EGF gave a high activity of maintenance of hepatocyte adhesion and function compared with soluble HB-EGF. Finally, when lowering temperature to 20°C, the cultured hepatocyte sheets were detached from the surface through the reduction of affinity binding between HE-EGF and immobilized heparin with increasing the mobility of heparin and steric hindrance of the swollen PIPAAm chains (Figure 1, right). Conclusions: Heparin-functionalized 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.