Three-dimensional circulation perfusion culture of hepatocytes in the liver decellularizedscaffold

Abstract

Objective: The intact rat liver decellularized scaffolds were preparedand repopulated hepatocytes by continuous perfusion technology.Toprovideexperimental support for the application of decellularized liver scaffolds in liver engineering. Method: Decellularized liver scaffolds were obtained by perfusing method. The composition and structure was examined by HE, Masson, Sirius red stain and immunofluorescence. The ultrastructure was examined by scanning electron microscope (SEM). DNA content was used to confirm the effect of decellularization. The circulation perfusion device was established. Hepatocytes were recellularized into the scaffolds by multiposition parenchymal injection method and infusion method, thenthe scaffolds were cultured in the circulation perfusion device in vitro. After cultivation, HE staining, immunofluorescence and SEM were conducted to observe the growth situation of hepatocytes in the scaffolds. Results: The rat decellularized liver scaffolds were successfully obtained by perfusion method. Histological staining demonstrated the remove of cellular component and the reservation of extracellular cellmatrix. Immunofluorescence staining demonstrated the retention of collagen I. SEM showed that the ultrastructure of the extracellular cell matrix presented thereticular structure. DNA content of the scaffolds was 47.5±18.1 ng/mg. The circulation perfusion device was composed of a peristaltic pump, oxygenator, chamber and the convey tubes. The multipositional parenchymal injection method resulted in a better engraftment rate. HE staining, immunofluorescence and SEM revealed that the growth and function of hepatocytes were goodin the scaffold. Conclusion: The decellularized rat liver scaffolds have favorable biochemical properties. The liver decellularized scaffolds applied with the circulation perfusion device could provide a well 3D plat form for culture of hepatocytes.