The Study On Decellularization Of Rat Liver And Recellularization Of Decellularized Rat Liver Scaffolds With Hepatocytes And EPCs

Objective To prepare decellularized scaffolds of rat liver and to establish a circulation perfusion device and replant rat hepatocytes and bone marrow-derived EPCs into the decellularized scaffolds respectively.And to provide experimental support for the vasculation of decellularized liver scaffolds in liver engineering.Methods(1)Decellularized rat liver scaffolds were obtained by perfusing method.The composition was detected by HE staining,Masson staining,sirius red stain and immunohistochemical staining.The ultrastructure was detected by scanning electron microscopy.DNA content was detected to confirm the effect of decellularization.GAG content was detected by ELISA.The toxicity of the decellularized scaffolds was detected by CCK-8.The scaffolds were tranplanted in the subcutaneous adipose layer on the back of heterologous animals to evaluate biocompatibility.(2)The circulation perfusion device was established.Hepatocytes were recellularized into the scaffolds by multiposition parenchymal injection method and infusion method by the circulation perfusion device in vitro.After cultivation,HE staining,immunofluorescence and scanning electron microscopy were used to observe the growth situation of hepatocytes in the scaffolds.Albumin and total bile acid secretion in the medium was detected to evaluate the liver function.(3)Bone marrow derived EPCs were characterized by immunofluorescence of CD31,CD133,DiI-ac-LDL and FITC-UEA.EPCs were repopulated into the decellularized scaffolds by portal-vein infusion method and cultured by the circulation perfusion device.HE staining and immunofluorescence of CD31 were conducted to observe the growth situation of EPCs in the scaffolds.Results(1)The decellulariezd rat liver scaffolds were successfully obtained by perfusion method.Histological staining showed cellular component was removed and extracellularmatrix was reserved.Immunohistochemical staining showed collagen I,collagen IV,fibronection and laminin were reserved.Scanning electron microscopy showed the ultrastructure of the extracellular matrix was presented.DNA content of the scaffolds were extremely low.GAG content was decreased.The scaffolds had no toxicity.Xeno-transplantation showed the scaffold had favourable biocompatibility.(2)The circulation perfusion device was composed of a peristaltic pump,chamber,oxygenator and the convey tubes.The multipositional parenchymal injection method resulted in a better engraftment rate.HE staining,immunofluorence and scanning electron microscopy revealed that hepatocytes grow well in the scaffold.(3)The primary cultured EPCs were postive for CD31,CD133,DiI-ac-LDL and FITC-UEA.HE staining and immunofluorence revealed that EPCs can be located around the blood vessel walls.conclusions(1)The decellularized rat liver scaffolds have favourable physical and chemical properties and biocompatibility.(2)The decellularized rat liver scaffolds provide a favourable 3D platform for the hepatocytes in the circulation perfusion device.(3)The primary cultured EPCs can grow around the blood vessel walls of the decellularized liver scaffolds by circulation perfusion method.