PE-009: 3D Printing of Mouse Primary Hepatocytes for Generating 3D Hepatic Structure

Purpose: Liver transplantation is the most clearly available treatment for severe liver disease. However, it is limited by donor organ shortage and donor pool. Recently, it is suggested that development of feasible technique isnecessary to overcome such limitation. Here, we suggest 3-dimentional (3D) bioprinting technique as one of the mostpromising techniques. Methods: To isolate mouse primary hepatocytes, collagenase was injected into 4-6 weeks mouse liver. Isolated hepatocytes were stained albumin, HNF4alpha and Hepar1 for confirming hepatocytes. Primary hepatocytes were mixed with 3% alginate and printed using 3D printer (made by KIMM). 3D printed hepatic structures were cultured with hepatocyte long term culture media, and its function and gene expression were conducted by qRT-PCR. To compare primary hepatocyte function, primary hepatocytes were also cultured bys and wich method and 2D. Sand wich cell culture used on a single surface was overlaid with a second layer of extracellular matrix, and 2D cell culture used on single surface was dry coating. cultured with hepatocyte long term culture media, and its function and gene expression were conducted by qRT-PCR. Results: We set up mouse liver perfusion system for isolating primary hepatocytes. Two-step colloagenase methodefficiently isolated primary hepatocytes (8X107 cells/mice). Our methods could isolate hepatocytes with 70~80% viability. These hepatocytes were immuno- stained and qRT-PCRwith albumin, CK18 for confirming functional hepatocytes. Isolated hepatocytes were highly expressed albumin and CK18 but not expressed AFP. To imitate functional liver organ,we made 3D hepatic structure (25×25mm) with primary hepatocytes using 3D bioprinter. Surprisingly, the cellswere survived more than 30 days in alginate structure without any morphological change as compare to collagen sandwich or 2D cultured cells. In addition to morphology of 3D printed hepatocytes, hepatic marker genes were still expressed. Conclusions: These results provide the methods for primary hepatocyte long-term culture and possibility of mimicking 3D liver structure. Also, this is suggesting a proof of in vivo-like morphology of a transplantable liver graft to be a potential treatment for liver disease.