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

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 4day 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. © 2016, The Japanese Society for Regenerative Medicine. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/ 4.0/). AAm, 2-carboxyisopropylacrylamide; TCPS, tissue culture polystyrene dishe; EGF, epidermal growth factor; bFGF, basic EGF-like growth factor; PIPAAm, poly(N-isopropylacrylamide) on TCPS; IC, poly(N-isopropylacrylamide-co-2eparin-modified IC; HB-EGFX/heparin-IC, HB-EGF-bound heparin-IC; PIPAAm þ HB-EGFY, PIPAAm with soluble HBdimetylaminopropyl)-carbodiimide hydrochloride; MES, morpholinoethanesulfonic acid monohydrate; ECM, extraedium; PBS, Dulbecco's phosphate buffered saline; EDTA, trypsin/ethylenediaminetetraacetic acid; FBS, fetal bovine r 4 alpha; F7, coagulation factor VII; F9, coagulation factor IX; ELISA, enzyme-linked immunosorbent assay; RT-PCR, 5; fax: þ81 3 3359 6046. 1; fax: þ81 3 3359 6046. bayashi), [email protected] (T. Okano). Society for Regenerative Medicine. ative Medicine. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license Y. Arisaka et al. / Regenerative Therapy 3 (2016) 97e106 98