Plastic Surgery Research Council
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PSRC 60th Annual Meeting
Program and Abstracts

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Free Flap as the Vascularized Foundation for Hepatic Tissue Engineering
Peter A. Than, MD, Christopher R. Davis, BSc MB ChB MRCS, Michael W. Findlay, MBBS PhD FRACS FACS, Wei Liu, MD PhD, Sacha M.L. Khong, PhD, Geoffrey C. Gurtner, MD FACS.
Stanford University School of Medicine, Stanford, CA, USA.

Purpose: Creation of patient-specific replacement organs promises to address major shortcomings of current strategies in transplantation, namely relating to the need for life-long immunosuppression and limited donor supply. We previously described a novel approach to fabricate autologous, vascularized neo-organs in vitro. This method uses expendable microcirculatory beds (EMBs) found within free flaps that are maintained ex vivo on a perfusion bioreactor and seeded with autologous stem cells. Unlike existing tissue engineering paradigms that rely upon vascular ingrowth from the wound bed, this method starts with the intact vascular system and surrounding stromal support and builds tissue from the “inside out.” In this fashion, we address the blood supply from the time of implantation, which is a critical bottleneck in current tissue engineering strategies. Here, building upon this we developed a protocol for the differentiation of autologous adipose-derived stem cells (ASCs) into hepatocyte-like cells in vitro, for the purpose of creating a functional neo-liver. We also demonstrate that tri-cultures recapitulating fetal hepatic developmental conditions result in spontaneous organization into hepatic buds.
Methods: ASCs were harvested from the inguinal fat pads of Wistar rats by collagenase digestion. The cells were expanded in culture in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS) and 1% Penicillin-Streptomycin (PSG). At passage 3, hepatocyte differentiation was begun. The initial media consisted of DMEM, 10% FBS, 1% PSG, Hepatocyte Growth Factor (HGF) 50 ng/mL, and Fibroblast Growth Factor (FGF) 10 ng/mL for 14 days. After this period, complete Williams’ media supplemented with 5% FBS, Dexamethasone, Insulin, Transferrin, Selenium, and Oncostatin M 30 ng/mL was used for 14 days. Differentiation was assessed by bright-field microscopy, immunohistochemistry, and western blot.
Co-culture experiments were performed using rat aortic endothelial cells (RAECs), ASCs, and hepatocytes. ASCs and RAECs were labeled with fluorescent cell tracking dye prior to co-seeding. 5x104 ASCs, 2x105 RAECs, and 5x104 hepatocytes were co-seeded into 24 well plates and cultured in a 1:1 mixture of EBM-2 and Williams’ media containing 10% FBS and 1% PSG. Organization into liver buds was monitored by bright-field microscopy, fluorescence microscopy, and immunohistochemistry.
Results: ASCs exposed to differentiation media demonstrated a distinct morphology change, developing a hepatocyte-like appearance by day 21. Albumin and cytochrome P450 production was confirmed by immunohistochemistry and western blot. Tri-cultured ASCs, RAECs, and hepatocytes spontaneously organized into 3-dimensional hepatic buds by day 1 in culture. Co-localization of all three cell types and hepatocyte function were confirmed by immunohistochemistry.
Conclusions: A successful protocol for the differentiation of rat ASCs into hepatocyte-like cells has been established, cementing a renewable supply of autologous liver replacement cells. We are able to efficiently maintain and seed EMBs with ASCs ex vivo. To further improve the rate and bulk of hepatic tissue formation, we demonstrate the ability to re-capitulate the hepatic developmental environment through co-seeding. These are critical steps in our approach to engineer an autologous neo-liver and address several important barriers in tissue engineering.


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