Islet Neogenesis-associated Protein Improves The Differentiation Of Islet-like Clusters From Human Pancreatic Duct Cells

Objective To explore the method of isolation, purification, cultivation and identification of adult human pancreatic duct cells in vitro.Methods The pancreastic tissue were digested by injecting collagenase P solution and purified by removing the islets with Ficoll density gradient centrifugation. The islet-depleted tissue was cultured in CMRL1066 with 10% fetal calf serum, then the cells could form a monolayer after 7-10 days. 0.25% Trypsin-EDTA was used to detach the cells, which were serial-subcultivation. Cells from passage(s) 2~6 were used to detect the expression of CK19, Pdx-1, Nestin, Insulin and Glucagon with methods of immunofluorescence staining and RT-PCR.Results Following collagenase solution digesting and purification on continuous Ficoll gradients, the pancreatic duct cells could get a better purification. The adherent cobblestone-like cells were expanded in monolayer from the duct cell aggregates, reaching 70-80% confluence after 7-10 days of culture. The results of immunofluorescence staining revealed that the expression of CK19, Pdx-1 and Nestin of cells were positive, and the rate of positive cells were (87.5±6.2)%, (77.5±8.6)% and (50.9±9.5)%, respectively, while Insulin staining were negative. The results of RT-PCR demomstrated that the cells also expressed CK19, Pdx-1 and Nestin. No Insulin or Glucagon expression was detected.Conclusions This method can isolate the pancreatic duct cells well, and the cells obtained have the character of stem cells through identification. Objective To explore the method of preparation of acellular islet matrix, and to determine its potential as a tissue-engineering scaffold.Methods The human pancreatic islets were treated sodium-dodecyl-sulphate alone or sodium-deoxycholate combined with tert-octylphenyl-polyoxyethylen followed by a washing process with M-199 medium to remove any residual detergents and an enzymatic digestion with deoxyribonuclease/ribonuclease to remove remaining nucleic acids. These 2 different decellularization protocols were compared in islets for efficiency of complete cell removal and matrix integrity by morphology and Immunofluorescent staining.Results After treatment with high concentration ( 0.1%, 0.05%) of sodium- dodecyl-sulphate,the cell components were removed uncompletely and unevenly, with strikingly destroying the underlying extracellular matrix structure, while no significant cell components removing could be detected within the most islets treated with 0.01% SDS. Compared with sodium-dodecyl-sulphate involving decellul -arization procedures, there were strikingly barbs arounding the whole islet in group 2 when finished the treatment with detergents. The decellularization protocol, based on 0.25% sodium-deoxycholate together with 0.01% tert-octylphenyl- polyoxyethylen, completely removed islet cell components and preserved matrix integrity. Free of any Immunofluorescent DAPI staining detectable corroborated completely cell removal while collagenⅠ,collagenⅢand elastin could be detected positive.Conclusions The combination of sodium-deoxycholate and TritonX-100 can be used as a better method in preparation of acellular islet matrix, which could be a good candidate scaffold for rebuilding the tissue engineering islet.