Bioengineering of a functional sheet of islet cells for the treatment of diabetes mellitus Hirofumi Shimizu a , Kazuo Ohashi b, * , Rie Utoh b , Kazuya Ise a , Mitsukazu Gotoh a , Masayuki Yamato b , Teruo Okano b a Department of Surgery 1, Fukushima Medical University, 1 Hikarigaoka, Fukushima-shi, Fukushima 960-1295, Japan b Institute of Advanced Biomedical Engineering and Science, Tokyo Women’s Medical University, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666, Japan article info Article history: Received 12 July 2009 Accepted 22 July 2009 Available online 11 August 2009 Keywords: Islet transplantation Cell sheet Laminin-5 Monolayer culture abstract The present study was designed to establish a novel tissue engineering approach for diabetes mellitus (DM) by fabricating a tissue sheet composed of pancreatic islet cells for in vivo transplantation. Pancreatic islet cell suspensions were obtained from Lewis rats, and plated onto temperature-responsive culture dishes coated with extracellular matrix (ECM) proteins. After the cells reached confluency, islet cells cultured on laminin-5 coated dishes were successfully harvested as a uniformly spread tissue sheet by lowering the culture temperature to 20  C for 20 min. The functional activity of the islet cell sheets was confirmed by histological examination and Insulin secretion assay prior to in vivo transplantation. Histological examination revealed that the harvested islet cell sheet was comprised of insulin- (76%) and glucagon- (19%) positive cells, respectively. In vivo functionality of the islet cell sheet was maintained even 7 days after transplantation into the subcutaneous space of Lewis rats. The present study describes an approach to generate a functional sheet of pancreatic islet cells on laminin-5 coated temperature- responsive dishes, which can be subsequently transplanted in vivo. This study serves as the foundation for the creation of a novel cell-based therapy for DM to provide patients an alternative method. Ó 2009 Elsevier Ltd. All rights reserved. 1. Introduction Cell-based therapy using pancreatic islets has been established as a promising new approach for treating insulin-dependent diabetes mellitus (DM). Compared to a whole organ pancreas transplantation, islet cell-based therapies offer advantages in terms of its minimal invasiveness as well as the need for only a short stay in the hospital following the procedure. In a recent international clinical trial, it was reported that 44% of the DM patients transplanted with islet cells for 1 year were found to successfully restore their insulin production and stably maintain their glycemic profile [1]. However, the func- tional viability of the transplanted islet cells was markedly decreased down to 14% following the 2 year time point after transplantation. To advance the islet-based therapies for DM, it is clear that further improvements are needed to optimize the conditions to maximize the longevity of the transplanted cell systems. Islet transplantation has been previously conducted using a procedure involving the injection of islet cells into the portal circulation leading to the liver. Several issues related with the islet transplantation via the portal circulation have remained unre- solved. An enormous amount of blood supply contributes to the maintaining the transplanted cells, and the secreted insulin requires ready access to the periphery of the portal branches in the liver. However, researchers have demonstrated that only a small fraction of transplanted islet cells can successfully engraft to the surrounding tissue due to several types of inflammatory-mediated immune responses mounted by the host species. This includes an instant blood mediated inflammatory reaction (IBMIR), activation by direct exposure to the foreign immunologic cells, and the toxic effects of the immunosuppressive compounds on the transplanted islets [2,3]. These immune-related responses appear to occur when the islets are physically located within the hepatic vasculature. To circumvent the immune system activation, several studies have attempted to transplant islet cells into extrahepatic sites, such as spleen [4], abdominal cavity [5], omentum [6], testis [7], and renal subcapsular space [8], and shown to have some modicum of success to regulate blood glucose levels. Other studies have attempted to transplant cells to extra-portal heterotopic sites using bio-engineered islet tissue constructs comprised of islets and biodegradable polymeric matrices [9], which would act as a mechanical barrier for the transplanted islet cells from activating the immune system [10,11]. Scaffold devices * Corresponding author. Tel.: þ81 3 3353 8111x66214; fax: þ81 3 3359 6046. E-mail address: ohashi@abmes.twmu.ac.jp (K. Ohashi). Contents lists available at ScienceDirect Biomaterials journal homepage: www.elsevier.com/locate/biomaterials 0142-9612/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2009.07.042 Biomaterials 30 (2009) 5943–5949