633. Reconstruction of Vascularized Subcutaneous Liver Tissue for Minimally Invasive Cell Therapy

Abstract

Subcutaneous liver tissue construction is an attractive and minimally invasive approach used to overcome the chronic shortage of donor livers for the curative treatment of hepatic failure. However, graft failure occurs frequently due to lack of blood vessels. In this study, we describe a subcutaneous human liver reconstruction approach allowing for rapidly vascularized grafts by transplanting engineered hepatocyte/fibroblast sheets (EHFSs).Human primary hepatocytes were isolated and purified from surgically resected human liver tissues by the collagenase perfusion method. EHFSs were fabricated using temperature-responsive culture dishes (TRCDs) (UpCell; CellSeed, Tokyo). The hepatocytes were plated onto a confluent layer of normal human dermal fibroblasts (TIG-118 cells). After 4 days in culture, EHFSs were harvested and transplanted subcutaneously into immunodeficient mice. The expression levels of angiogenic factors in vitro, liver-specific functions and structures in vivo, and therapeutic potential of serious liver diseases were evaluated.EHFSs exhibited significantly higher synthesis rates of VEGF, TGF-b1, and HGF than the hepatocyte-only sheets (HSs) from 1 to 3 days in culture. This result indicate the possibility of using a one-step subcutaneous hepatocyte transplantation procedure without a pre-transplant vascularization step such as implanting a FGF-releasing device. The transplanted EHFSs were laminated and formed a vascularized subcutaneous human liver tissues (VSLTs). The VSLTs had liver-specific features such as glycogen stores and albumin, A1AT, and coagulation factor IX syntheses. The aggregated hepatocytes formed several linear structures. Furthermore, subacute hepatic failure model mice (TK-NOG mice) with VSLT were alive at least 7 weeks after liver damage.The present study describes a new approach for vascularized human liver organogenesis under mouse skin. This approach could prove valuable for establishing novel minimally invasive cell therapies for liver diseases.