title: Heparin-gelatin mixture improves vascular reconstruction efficiency and hepatic function in bioengineered livers.
abstract: Whole organ decellularization is a cell removal process that creates a natural extracellular matrix for use in transplantation. A lack of an intact endothelial layer in the vascular network of decellularized organs results in blood clotting even with anti-coagulation treatment. Furthermore, shear stress caused by blood flow may affect reseeded parenchymal cells. We hypothesized that a heparin-gelatin mixture (HG) can act as an antithrombotic coating reagent and induce attachment and migration of endothelial cells (ECs) on vascular wall surfaces within decellularized livers, with subsequent parenchymal cell function enhancement. Portal vein (PV) perfusion was performed for right lateral lobe decellularization of porcine livers. We tested if HG-precoating of isolated decellularized PV could increase EC attachment and migration. Additionally, we coated PV and hepatic artery walls in decellularized liver with HG, and then repopulated it with ECs and maintained it under vascular flow in a bioreactor for 10days. Re-endothelialized scaffolds were perfused with porcine blood for thrombogenicity evaluation. We then co-cultured hepatocellular carcinoma (HepG2) cells and ECs to evaluate the effect of endothelialization on parenchymal cells. Finally, we transplanted these scaffolds heterotopically in pigs. HG improved ECs' ability to migrate and adhere to vessel discs. ECs efficiently covered the vascular compartments within decellularized scaffolds and maintained function and proliferation after HG-precoating. No thrombosis was observed after 24h blood perfusion in HG-precoated scaffolds, indicating an efficiently endothelialized vascular tree. HepG2 cells displayed a higher function in scaffolds endothelialized after HG-precoating compared to uncoated scaffolds in vitro and after in vivo transplantation. Our results lay the groundwork for engineering human-sized whole-liver scaffolds for clinical applications.
  
  
  
  A major obstacle to successful organ bioengineering is vasculature reconstruction to avoid thrombosis and deliver nutrients through blood to the whole scaffold after in vivo transplantation. Although many attempts have been made to construct endothelial cell layers on the vascular network within decellularized organs, complete coverage has not be achieved. Here, we describe an effective approach for endothelial cell seeding to reconstruct a patent vascular tree within decellularized livers by coating the vasculature using heparin-gelatin mixture. Our results have demonstrate that enhancement of endothelial cell attachment by heparin-gelatin treatment could improve vascular patency and parenchymal cell function in vitro and in vivo. These results represent a significant advancement toward bioengineering functional liver tissue that maintains vascular patency for transplantation.
authors.affiliation: Stem Cell Institute, Kangwon National University, Chuncheon, Gangwon 24341, Republic of Korea; Research Institute for Veterinary Science, College of Veterinary Medicine, Seoul National University, Seoul 08826, Republic of Korea; Adult Stem Cell Research Center, College of Veterinary Medicine, Seoul National University, Seoul 08826, Republic of Korea. Electronic address: kamalhussein1986@gmail.com.
authors.forename: Kamal Hany
authors.initials: KH
authors.lastname: Hussein
authors.affiliation: Stem Cell Institute, Kangwon National University, Chuncheon, Gangwon 24341, Republic of Korea; Research Institute for Veterinary Science, College of Veterinary Medicine, Seoul National University, Seoul 08826, Republic of Korea; Adult Stem Cell Research Center, College of Veterinary Medicine, Seoul National University, Seoul 08826, Republic of Korea. Electronic address: vethouse2000@gmail.com.
authors.forename: Kyung-Mee
authors.initials: KM
authors.lastname: Park
authors.affiliation: Research Institute for Veterinary Science, College of Veterinary Medicine, Seoul National University, Seoul 08826, Republic of Korea; Adult Stem Cell Research Center, College of Veterinary Medicine, Seoul National University, Seoul 08826, Republic of Korea. Electronic address: kangpub@snu.ac.kr.
authors.forename: Kyung-Sun
authors.initials: KS
authors.lastname: Kang
authors.affiliation: Stem Cell Institute, Kangwon National University, Chuncheon, Gangwon 24341, Republic of Korea; College of Veterinary Medicine, Kangwon National University, Chuncheon, Gangwon 24341, Republic of Korea. Electronic address: woohm@kangwon.ac.kr.
authors.forename: Heung-Myong
authors.initials: HM
authors.lastname: Woo
mesh_terms: D000818:Animals; D057005:Bioengineering; D018920:Coculture Techniques; D042783:Endothelial Cells / Q000378:metabolism*; D005780:Gelatin / Q000737:chemistry*; D056945:Hep G2 Cells; D006493:Heparin / Q000737:chemistry*; D006801:Humans; D019164:Liver, Artificial; D018919:Neovascularization, Physiologic; D013552:Swine
chemical_list: D005780:Gelatin; D006493:Heparin
keywords: Decellularized livers; Endothelialization; Liver tissue engineering; Transplantation; Vascular reconstruction