IN SITU METHOD FOR HEPATIC DECELLULARIZATION IN MICE PRODUCES BIOCOMPATIBLE SCAFFOLDS FOR RECELLULARIZATION

Abstract

<h3>Background</h3> The production of a natural scaffold that mimics the native liver, provides structural support and molecular signals ideal for cells, plays a crucial role in liver tissue engineering and is being constantly investigated. Therefore, in this study we developed a simple and in situ method for hepatic decellularization in C57BL6 mice. <h3>Methods</h3> A 26G catheter was inserted into the caudal vena cava (CVC) and 50ml of 1X phosphate-buffered saline (PBS) containing 12.5U / ml of heparin was perfused, followed by 0.5% sodium dodecyl sulfate (SDS) for 4 hours, and PBS for 1 hour (infusion rate of 6ml / min). The liver was removed from the abdominal cavity and 50 ml of 0.1% peracetic acid in a 4% ethanol solution was infused for sterilization. The hepatic lobes were sectioned in 0.5 cm fragments and kept in culture medium for 4 days. AML12 hepatic cell lineage and primary hepatocytes were co-cultured with scaffolds for 14 days to verify biocompatibility. Histological analyzes, scanning electron microscopy (SEM), quantification of residual DNA, immunohistochemistry, microbiological analyzes of sterility and presence of mycoplasmas by PCR were performed to characterize the hepatic scaffold. <h3>Results</h3> After six hours of decellularization process, the hepatic ECM was translucent and the vascularization was preserved. As a plus in this decellularization protocol, the kidneys were also decellularized. Cell removal was observed in H&E staining and by DAPI fluorescence. The residual DNA was <50ng/dry weight. Positive immunostaining was observed for fibronectin, laminin, collagen I, III and V in decellularized matrix. The preservation of collagen fibers and glycosaminoglycans were confirmed by picrosirius red and alcian blue staining, respectively. The scaffold‘s sterility was also confirmed. <h3>Conclusion</h3> Our results present a promising new methodology for hepatic decellularization in small animal models, such as the mouse, which was biocompatible for cell culture aiming the recellularization process. Suporte: CAPES, CNPq, FAPESP.