A heparin-modified thermoresponsive surface with heparin-binding epidermal growth factor-like growth factor for maintaining hepatic functions

Autor: Yoshinori, Arisaka, Jun, Kobayashi, Kazuo, Ohashi, Kohei, Tatsumi, Kyungsook, Kim, Yoshikatsu, Akiyama, Masayuki, Yamato, Teruo, Okano
Rok vydání: 2015
Předmět:
Alb
albumin
IPAAm
N-isopropylacrylamide
F7
coagulation factor VII
CIPAAm
2-carboxyisopropylacrylamide
TCPS
tissue culture polystyrene dishe
PBS
Dulbecco's phosphate buffered saline
bFGF
basic fibroblast growth factor
RT-PCR
reverse transcription polymerase chain reaction
FBS
fetal bovine serum
IC
poly(N-isopropylacrylamide-co-2-carboxyisopropylacrylamide) on TCPS
Poly(N-isopropylacrylamide)
EDC
1-ethyl-3-(3-dimetylaminopropyl)-carbodiimide hydrochloride
EGF
epidermal growth factor
DMEM
Dulbecco's modified Eagle's medium
EDTA
trypsin/ethylenediaminetetraacetic acid
HB-EGFX/heparin-IC
HB-EGF-bound heparin-IC
Heparin
MES
morpholinoethanesulfonic acid monohydrate
ELISA
enzyme-linked immunosorbent assay
NHS
N-hydroxysuccinimide
heparin-IC
heparin-modified IC
ECM
extracellular matrix
Hnf4α
hepatocyte nuclear factor 4 alpha
F9
coagulation factor IX
Thermoresponsive cell culture surface
HB-EGF
heparin-binding EGF-like growth factor
Hepatocyte sheet
PIPAAm + HB-EGFY
PIPAAm with soluble HB-EGF
Original Article
Heparin-binding EGF-like growth factor
PIPAAm
poly(N-isopropylacrylamide) on TCPS
hormones
hormone substitutes
and hormone antagonists
Zdroj: Regenerative Therapy
ISSN: 2352-3204
Popis: 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.
Graphical abstract
Highlights • Heparin-modified thermoresponsive surfaces bound with HB-EGF were designed. • HB-EGF bound on the heparinized surface maintained the function of hepatocytes. • Hepatocyte sheets were detached from the surface only after lowering of the temperature to 20 °C. • The heparinized thermoresponsive surface can be used to fabricate transferrable hepatocyte sheets.
Databáze: OpenAIRE
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