A heparin-modified thermoresponsive surface with heparin-binding epidermal growth factor-like growth factor for maintaining hepatic functions in vitro and harvesting hepatocyte sheets

Abstract

A heparin-modified thermoresponsive surface bound with heparin-binding epidermal growth factor-like growth factor (HB-EGF) was designed to allow creation of transferrable and functional hepatocyte sheets. A heparin-modified thermoresponsive surface was prepared by covalently tethering heparin onto poly( N -isopropylacrylamide- co -2-carboxyisopropylacrylamide)-grafted tissue culture polystyrene surfaces (Heparin-IC). HB-EGFs were able to stably bind to heparin-IC via affinity interaction. The survival of primary rat hepatocytes was maintained through HB-EGF-bound heparin-IC (HB-EGF/heparin-IC). Moreover, cultured rat primary hepatocytes on HB-EGF/heparin-IC exhibited higher albumin-secretion than hepatocytes cultured on PIPAAm-grafted and collagen-coated surfaces with soluble HB-EGF in the culture medium, regardless of whether soluble EGF was added. These results suggested that HB-EGF/heparin-IC is able to effectively maintain hepatic function via continuous signaling of HB-EGF. After a 4-day cultivation, the cultured hepatocytes on HB-EGF/heparin-IC detached as a cell sheet with fibronectin and HB-EGF only after the temperature was lowered to 20 °C. In addition, higher expression of hepatocyte-specific genes (albumin, hepatocyte nuclear factor 4 alpha, coagulation factor VII, and coagulation factor IX) in hepatocyte sheets was detected on HB-EGF/heparin-IC than on a PIPAAm surface with soluble HB-EGF, indicating that HB-EGF/heparin-IC suppressed the dedifferentiation of cultured hepatocytes. Hence, heparin-modified thermoresponsive surfaces bound with HB-EGF facilitate the fabrication of transferrable hepatocyte sheets with intact hepatic functions and have the potential to provide an in vitro culture system using functional hepatocyte sheet tissues, which may serve as an effective hepatocyte-based tissue engineering platform for liver disease treatments.

Cell sheet tissue engineering: Cell sheet preparation, harvesting/manipulation, and transplantation

“…Recently, our group utilized a heparin‐immobilized poly(IPAAm‐ co ‐CIPAAm) surface [Fig. (C)] to bind heparin‐binding proteins such as basic fibroblast growth factor (bFGF or FGF‐2) and heparin‐binding epidermal growth factor‐like growth factor (HB‐EGF) . For example, bFGF bounds with heparin with high affinity ( K D = 3.9 × 10 −8 M ) and also reinforced the binding with bFGF and FGF receptor (FGFR) through the formation of a heparin/bFGF/FGFR complex [Fig.…”

“…49 HB-EGF-bound heparin-immobilized temperature-responsive surfaces facilitate the fabrication of hepatocyte sheets with preserved hepatic functions. 48 Tissue engineering of the liver is an attractive approach for the treatment of liver diseases such as enzyme deficiencies and hemophilia. 54 Engrafted hepatocyte sheets in a pre-vascularized subcutaneous site exhibited long-term secretion of liver-specific enzymes.…”

Design of Temperature-Responsive Polymer-Grafted Surfaces for Cell Sheet Preparation and Manipulation

“…The utilization of an HB-EGF-bound heparin-immobilized PIPAAm-grafted surface enables hepatocytes to be cultured while maintaining hepatic functions and to be recovered as a contiguous sheet only by changing the temperature. 28 The number of cultured hepatocytes on the HB-EGF-bound surface is constant over 4 days, whereas the number is dramatically decreased under the conditions of culture systems without HB-EGF. Therefore, stimulation of the EGF receptor is essential for the in vitro survival of hepatocytes.…”

“…The heparin-immobilized PIPAAm-grafted surface facilitates the incorporation of heparin-binding proteins via affinity. We reported heparin-immobilized PIPAAm-grafted surfaces bound with basic fibroblast growth factor (bFGF, or FGF-2) 27 and heparin-binding epidermal growth factor-like growth factor (HB-EGF) 28 for the acceleration of fibroblast growth and the preservation of hepatic functions, respectively.…”

Biomaterials for Sequestration of Growth Factors and Modulation of Cell Behavior

“…Additionally, by simply lowering the temperature to 20 °C after 4 day cultivation, it was possible to detach a cell sheet which retained fibronectin and EGF. [108] Another method of producing hepatocyte cell sheets was proposed by Hwang et al [109] A heparin-based hydrogel was micropatterned on a polyelectrolyte multilayer using photopolymerization and lithographic techniques. The system could precisely release a combination of HGF, EGF, and FGF, inducing ASC differentiation into the hepatic lineage.…”