Date: Sunday, May 3, 2015
Session Time: 5:30pm-6:30pm
Presentation Time: 5:30pm-6:30pm
Location: Exhibit Hall E
Background: Creation of patient-specific replacement organs promises to address major shortcomings in transplantation. We previously described an approach to fabricate autologous, vascularized neo-organs by using expendable microcirculatory beds (EMBs) found within free flaps that are maintained ex vivo on a perfusion bioreactor and seeded with stem cells. Unlike tissue engineering paradigms that rely upon vascular ingrowth from the wound bed, this method starts with the intact vascular system and builds tissue from the inside out. Applying this to liver engineering, we developed a protocol for hepatic differentiation of autologous adipose-derived stem cells (ASCs). We also demonstrate that tri-culture recapitulates fetal hepatic developmental conditions resulting in spontaneous organization into hepatic buds.
Methods: ASCs were harvested from the inguinal fat of Wistar rats and expanded in culture. Hepatic differentiation was begun using Dulbeccos Modified Eagle Medium (DMEM), Fetal Bovine Serum (FBS), Hepatocyte Growth Factor (HGF), and Fibroblast Growth Factor (FGF) for 14 days. After this period, complete Williams media supplemented with FBS, Dexamethasone, Insulin, Transferrin, Selenium, and Oncostatin M was used for 14 days.
For co-culture experiments, rat aortic endothelial cells (RAECs) and ASCs were labeled with fluorescent cell tracking dye, tri-seeded with hepatocytes into 24 well plates, and cultured in a 1:1 mixture of EBM-2 and Williams media. Differentiation and hepatic function were assessed by bright-field microscopy, fluorescence microscopy, immunohistochemistry, and western blot.
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.
Conclusion: Hepatic differentiation of ASCs cements a renewable supply of autologous liver replacement cells. To further improve the rate and bulk of hepatic tissue formation, we demonstrate the ability to recapitulate the liver developmental environment through co-seeding. These advances, combined with our EMB-based tissue engineering approach, address critical barriers in liver engineering.
To cite this abstract in AMA style:Than P, Davis C, Findlay M, Liu W, Le T, Khong S, Melcher M, Gurtner G. Inside Out Autologous Neo-Liver Fabrication [abstract]. Am J Transplant. 2015; 15 (suppl 3). https://atcmeetingabstracts.com/abstract/inside-out-autologous-neo-liver-fabrication/. Accessed November 21, 2019.
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