Objective To seek an appropriate culture condition in which porcine hepatocytes can continuously proliferate and express their normal differentiation phenotypes for a longer period of time. Methods Different types a...Objective To seek an appropriate culture condition in which porcine hepatocytes can continuously proliferate and express their normal differentiation phenotypes for a longer period of time. Methods Different types and dosages of reagents,transferrin,epidermal growth factor and nicotinamide were used to culture porcine hepatocytes in serum-free Dulbecco’s modified Eagle’s medium (DMEM). And the proliferative effects and functions of the cells were detected at different culture times. Results Transferrin at 5 ng/ml,nicotinamide at 10 mmol/L and epidermal growth factor at 10 ng/ml had better effects on the viability of hepatocytes than DMEM,DMEM+10% fetal cattle serum FCS and other dosages of these reagents ( P <0.05). OD values of MTS were still high in culture at day 7 and day 10,while nearly 30%-35% cells went into the S phase. Good hepatocyte funcitons were found in these groups,and the secretion of albumin was positively correlated with OD value of MTS. The levels of aspartate transaminase and ammonia in these media were lower than those in DMEM and DMEM+FCS.Conclusion Transferrin at 5 ng/ml,epidermal growth factor at 10 ng/ml and nicotinamide at 10 mmol/L are benefitial to the viability and proliferation of hepatocytes.展开更多
AIM:Clinical application of human hepatocytes (HC) is hampered by the progressive loss of growth and differentiation in vitro. The object of the study was to evaluate the effect of a biphasic culture technique on expr...AIM:Clinical application of human hepatocytes (HC) is hampered by the progressive loss of growth and differentiation in vitro. The object of the study was to evaluate the effect of a biphasic culture technique on expression and activation of growth factor receptors and differentiation of human adult HC. METHODS: Isolated HC were sequentially cultured in a hormone enriched differentiation medium (DM) containing nicotinamide, insulin, transferrin, selenium, and dexame-thasone or activation medium (AM) containing hepatocyte growth factor (HGF), epidermal growth factor (EGF), and granulocyte-macrophage colony-stimulating factor (GMCSF). Expression, distribution and activation of the HC receptors (MET and EGFR) and the pattern of characteristic cytokeratin (CK) filaments were measured by fluorometry, confocal microscopy and Western blotting. RESULTS: In the biphasic culture system, HC underwent repeated cycles of activation (characterized by expression and activation of growth factor receptors) and re-differentiation (illustrated by distribution of typical filaments CK-18 but low or absent expression of CK-19). In AM increased expression of MET and EGFR was associated with receptor translocation into the cytoplasm and induction of atypical CK-19. In DM low expression of MET and EGFR was localized on the cell membrane and CK-19 was reduced. Receptor phosphorylation required embedding of HC in collagen type I gel. CONCLUSION: Control and reversible modulation of growth factor receptor activation of mature human HC can be accomplished in vitro, when defined signals from the extracellular matrix and sequential growth stimuli are provided. The biphasic technique helps overcome dedifferentiation, which occurs during continuous stimulation by means of growth factors.展开更多
文摘Objective To seek an appropriate culture condition in which porcine hepatocytes can continuously proliferate and express their normal differentiation phenotypes for a longer period of time. Methods Different types and dosages of reagents,transferrin,epidermal growth factor and nicotinamide were used to culture porcine hepatocytes in serum-free Dulbecco’s modified Eagle’s medium (DMEM). And the proliferative effects and functions of the cells were detected at different culture times. Results Transferrin at 5 ng/ml,nicotinamide at 10 mmol/L and epidermal growth factor at 10 ng/ml had better effects on the viability of hepatocytes than DMEM,DMEM+10% fetal cattle serum FCS and other dosages of these reagents ( P <0.05). OD values of MTS were still high in culture at day 7 and day 10,while nearly 30%-35% cells went into the S phase. Good hepatocyte funcitons were found in these groups,and the secretion of albumin was positively correlated with OD value of MTS. The levels of aspartate transaminase and ammonia in these media were lower than those in DMEM and DMEM+FCS.Conclusion Transferrin at 5 ng/ml,epidermal growth factor at 10 ng/ml and nicotinamide at 10 mmol/L are benefitial to the viability and proliferation of hepatocytes.
基金Supported by the "Matthias Lackas-Stiftung", "Paul und Ursula Klein-Stiftung", "Heinrich und Erna Schaufler-Stiftung", "Gisela Stadelmann-Stiftung", and study grants from the Johann Wolfgang Goethe-Universitatsklinikum,Universitatsklinikum Essen (IFORES),and Deutsche Forschungsgemeinschaft (AU 117/4-1)
文摘AIM:Clinical application of human hepatocytes (HC) is hampered by the progressive loss of growth and differentiation in vitro. The object of the study was to evaluate the effect of a biphasic culture technique on expression and activation of growth factor receptors and differentiation of human adult HC. METHODS: Isolated HC were sequentially cultured in a hormone enriched differentiation medium (DM) containing nicotinamide, insulin, transferrin, selenium, and dexame-thasone or activation medium (AM) containing hepatocyte growth factor (HGF), epidermal growth factor (EGF), and granulocyte-macrophage colony-stimulating factor (GMCSF). Expression, distribution and activation of the HC receptors (MET and EGFR) and the pattern of characteristic cytokeratin (CK) filaments were measured by fluorometry, confocal microscopy and Western blotting. RESULTS: In the biphasic culture system, HC underwent repeated cycles of activation (characterized by expression and activation of growth factor receptors) and re-differentiation (illustrated by distribution of typical filaments CK-18 but low or absent expression of CK-19). In AM increased expression of MET and EGFR was associated with receptor translocation into the cytoplasm and induction of atypical CK-19. In DM low expression of MET and EGFR was localized on the cell membrane and CK-19 was reduced. Receptor phosphorylation required embedding of HC in collagen type I gel. CONCLUSION: Control and reversible modulation of growth factor receptor activation of mature human HC can be accomplished in vitro, when defined signals from the extracellular matrix and sequential growth stimuli are provided. The biphasic technique helps overcome dedifferentiation, which occurs during continuous stimulation by means of growth factors.
