Purpose. To counterbalance the restricted availability of pancreatic islet tissue for transplant in Type 1 Diabetes Mellitus (T1DM), a new approach to enhance in vitro viability and function of isolated neonatal pancreatic porcine cell clusters (NPCC’s) is proposed by co-culturing them with PLGA microsphere (MS) entrapped, slowly release superoxide dismutase (SOD) and catalase (CAT). Methods. PLGA and PLA polymers were employed for protein encapsulation. A modified W/O/W double emulsion method was used for MS preparation and PEG400 was co-encapsulated for protein stabilization. Size, morphology, in vitro release and in vitro activity were assessed. For this purpose, Micro-BCA protein assay, UV, SEM, particle sizing techniques were employed. In vitro activity retention within MS was evaluated by simple NADH oxidation and H2O2 consumption assays. NPCCs viability was assessed at day 9 by staining with ethidium bromide and fluorescein-diacetate under fluorescence microscope, islet function was tested by static incubation with glucose and insulin was tested by radioimmunoassay. Real time polymerase chain reaction(RT-PCR) was performed for insulin and glut-2 rRNA determination. NPCC’s morphology was examined by optical microscopy upon staining with 1% osmium tetroxide (OsO4). Results. These powerful antioxidizing agents were shown to significantly improve morphology, viability and function, as assessed by microscopy, molecular, biochemical and functional studies, of the incubated NPCC’s, as compared to control. In fact, either static incubation or RNA analysis showed an increased output of insulin for NPCC’s treated with SOD and CAT, indicating a larger number of mature â-cells. Conclusion. Preliminarily, in vitro exposure of isolated NPCC’s to slow release microsphere-embedded SOD and CAT could permit or contribute to overcome hurdles associated with scarcity in islet tissue procurement for transplant in T1DM.
Long-term delivery of superoxide dismutase and catalase entrapped in poly(lactide-co-glycolide) microspheres: in vitro effects on isolated neonatal porcine pancreatic cell clusters.
GIOVAGNOLI, Stefano;LUCA, Giovanni;BLASI, PAOLO;RICCI, Maurizio;CALVITTI, Mario;CALAFIORE, Riccardo;ROSSI, Carlo
2005
Abstract
Purpose. To counterbalance the restricted availability of pancreatic islet tissue for transplant in Type 1 Diabetes Mellitus (T1DM), a new approach to enhance in vitro viability and function of isolated neonatal pancreatic porcine cell clusters (NPCC’s) is proposed by co-culturing them with PLGA microsphere (MS) entrapped, slowly release superoxide dismutase (SOD) and catalase (CAT). Methods. PLGA and PLA polymers were employed for protein encapsulation. A modified W/O/W double emulsion method was used for MS preparation and PEG400 was co-encapsulated for protein stabilization. Size, morphology, in vitro release and in vitro activity were assessed. For this purpose, Micro-BCA protein assay, UV, SEM, particle sizing techniques were employed. In vitro activity retention within MS was evaluated by simple NADH oxidation and H2O2 consumption assays. NPCCs viability was assessed at day 9 by staining with ethidium bromide and fluorescein-diacetate under fluorescence microscope, islet function was tested by static incubation with glucose and insulin was tested by radioimmunoassay. Real time polymerase chain reaction(RT-PCR) was performed for insulin and glut-2 rRNA determination. NPCC’s morphology was examined by optical microscopy upon staining with 1% osmium tetroxide (OsO4). Results. These powerful antioxidizing agents were shown to significantly improve morphology, viability and function, as assessed by microscopy, molecular, biochemical and functional studies, of the incubated NPCC’s, as compared to control. In fact, either static incubation or RNA analysis showed an increased output of insulin for NPCC’s treated with SOD and CAT, indicating a larger number of mature â-cells. Conclusion. Preliminarily, in vitro exposure of isolated NPCC’s to slow release microsphere-embedded SOD and CAT could permit or contribute to overcome hurdles associated with scarcity in islet tissue procurement for transplant in T1DM.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
