Figure 6
Nanostructured substrates preserve β-cell function in long term cultures. (A) Insulin granules density and distribution were evaluated by double immunofluorescence stainings with anti-insulin (red) and anti-chromogranin A (green) antibodies. The yellow-orange staining indicates colocalization between the two markers. Representative confocal images are shown. Bar = 5 μm. (B) The insulin content was evaluated in human islets cultured for 20 days on the indicated substrates or in freshly-isolated intact islets (three days old islets) (islets, black bars). Data (mean ± SD) are expressed as μUi of insulin per mg of protein (n = 3, in duplicate) (*p < 0.05; vs gelatin). (C) The insulin secretory response in basal (3.3 mM glucose) and stimulated (16.7 mM glucose) conditions was evaluated in human islets cultured for 20 days on the indicated substrates or in freshly isolated intact islets. Data (mean ± SD) are expressed as % of insulin content (n = 3, in duplicate). (*p < 0.05, vs gelatin; °p < 0.05, vs flat-ZrO2; ##p < 0.001 stimulated vs relative basal). (D) The cells were loaded with the Ca2+ indicator Fluo3 and Calcium imaging was performed under basal (NG, 3.3 mM glucose) (dotted lines) and stimulated (HG, 20 mM glucose) (continue lines) conditions. The time course of changes in fluorescence signals (F/F0) induced by glucose application (bars over traces) were recorded from islets grown on gelatin (black), flat-ZrO2 (blue) and ns-ZrOx (red). The curves illustrate average responses ± S.E. from four different islet isolations (N = 20 cells for each experiment) (p < 0.005 ns-ZrOx vs gelatin; P < 0.0001 ns-ZrOx vs flat-ZrO2). (E) Area Under the Curves (AUC) of experiments reported in D (*p < 0.05 ns-ZrOx vs gelatin; °°°p < 0.001 ns-ZrOx vs flat-ZrO2).
