The Experiment Of Regenerating A Insulin Secreting Organ By Decellularized Pancreatic Scaffold

Objective: To study the manufacture of decellularized pancreatic scaffold.To observe the regenerative condition of the MIN6 cells cultured in the 3D decellularized pancreatic scaffold.And to discuss the way in which the tissue enginneered pancreas played in restoring the function of insulin secretion.Method:(1).A laparotomy was performed and a 24 G catheter was inserted approximately 1 cm into the hepatic portal vein and sutured in place.The decellularized pancreatic scaffold was manufactured by means of perfusion.HE staining,scanning electron microscopy,DNA quantification and immunofluorescence were performed.In order to confirm the intact vasculature of the decellularized pancreatic scaffold,Trypan blue stain was infused into the portal vein to perform angiography.In vivo implantation of the decellularized pancreatic scaffold was performed to evaluate the biocompatibility.(2).MIN6 cells were cultured in the decellularized pancreatic scaffold by continous perfusion of culture medium.After five days of culture,the recellularized pancreatic scaffold was evaluated by HE staining,SEM analysis and immunohistochemistry.qRT-PCR was conducted for the functional gene of INS1 and INS2 to further illuminate the function of the MIN6 cells cultured in the 3D bioscaffold.(3).The recellularized pancreatic scaffold was implanted subcutaneously to the dorsal side of the diabetic mice.Fasting blood glucose was monitored to evaluate the function of the recellularized pancreatic scaffold in vivo.Result:(1).HE staining;SEM and DNA quantification of the decellularized pancreatic scaffold confirmed that there was little nucleic material left.Immunofluorescence of collagen 1 showed the retaining of an intact network of ECM after the whole process of decellularization.Angiography showed that the vasculature was reserved.In vivo implantation demonstrated the biocompatibility of the decellularized pancreatic scaffold.(2).After 5 days of culture,HE staining and SEM confirmed that MIN6 cells grew well in the scaffold.Immunohistochemistry and qRT-PCR further demonstrated the function of the cells grown in the scaffold was better than cells cultured on the culture dish.(3).Fasting blood glucose showed that the recellularized pancreatic scaffold can control the blood glucose of the diabetic mice and function well in vivo.Conclusion:(1).Perfusion based decellularization can effectively manufacture the decellularized pancreatic scaffold.(2).Decellularized pancreatic scaffold can support the growth and function of MIN6 cells.(3).The recellularized pancreatic scaffold can reduce the blood glucose of the dtabetic mice and it shed light on the new theraputic method of type 1 diabetes.