Biochemical Engineering Journal 52 (2010) 276–281 Contents lists available at ScienceDirect Biochemical Engineering Journal journal homepage: www.elsevier.com/locate/bej Development of hepatocyte-embedded hydrogel-filled macroporous scaffold cultures using transglutaminase Hiroyuki Ijima ∗ , Yung-Te Hou, Takayuki Takei Department of Chemical Engineering, Faculty of Engineering, Graduate School, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395, Japan article info Article history: Received 9 April 2010 Received in revised form 30 August 2010 Accepted 4 September 2010 Keywords: Animal cell culture Biomedical Immobilization Liver tissue engineering Tissue cell culture Transglutaminase abstract We aimed to develop cell-embedded gel-filled macroporous scaffold (CGS) culture technology as a funda- mental technology for the construction of large-scale tissue-like structures, which will be indispensable for practical liver tissue engineering. Tissue transglutaminase (tTGase) from guinea pig liver showed no cytotoxicity toward primary hepatocytes, and hepatocyte-embedded gelatin gel cultures could be real- ized. The albumin production activities in tTGase-mediated hepatocyte-embedded gelatin gel cultures were similar to those in collagen gel cultures, representing an established hepatocyte culture method for the expression of liver-specific functions. Therefore, it was expected that a hepatocyte-embedded gelatin gel culture system enabling high liver-specific function expression could be created. The CGS cul- ture system was created by incubating a suspension of hepatocytes and tTGase-containing gelatin-filled hydrophilic-treated scaffolds at 37 ◦ C. The albumin production activities in CGS with poly(l-lactic acid) macroporous scaffold (porosity = 68.4%) were inferior to those in tTGase–gelatin gel cultures because of a diffusion problem. However, the activities were similar between CGS with macroporous polyurethane foam (porosity = 98.8%) and tTGase–gelatin gel cultures, even under stationary conditions. On the other hand, further functional improvements of hepatocytes were achieved in HGF- or HGF/heparin-containing gel cultures. Based on these results, tTGase-mediated CGS cultures are expected to become a fundamental technology for the creation of engineered liver tissues. Crown Copyright © 2010 Published by Elsevier B.V. All rights reserved. 1. Introduction The liver is a central organ for metabolism in the body. There- fore, the creation of reconstruction technology for functional liver tissue equivalents using tissue engineering concepts is earnestly desired to save severe liver failure patients. However, the liver is a huge internal organ, and is difficult to grow in vitro. Furthermore, for the various functions and high oxygen requirement, good mass transfer is essential for the survival of hepatocytes. Therefore, the development of liver tissue engineering (LTE) technology is consid- ered to be one of the most difficult and important fields in tissue engineering. Recently, it was reported that the numbers of remaining trans- planted hepatocytes in prevascularized scaffolds were 6–18 times higher than those in control scaffolds [1]. However, practical appli- cation was impossible because the hepatocyte-occupied ratios were only 5.6–7.2% [1]. On the other hand, sheets of hepatic tis- sue have been transplanted into the subcutaneous space, and the transplanted cells were observed to form two-dimensional hepatic ∗ Corresponding author. Tel.: +81 92 802 2758; fax: +81 92 802 2758. E-mail address: ijima@chem-eng.kyushu-u.ac.jp (H. Ijima). tissues that stably persisted for longer than 200 days [2]. Upsizing and easy process development will be indispensable for practical use in the future [3]. On the other hand, the formation of tissue-like structures such as spheroids is effective for long-term survival and functional improvement of hepatocytes [4]. Furthermore, addition of growth factors is effective for the construction of functional liver tissue equivalents [5]. The functionality is more markedly improved by extracellular matrix (ECM)-derived hydrogel cultures [6,7]. In other words, all of the following aspects are indispensable for the real- ization of LTE: (i) cell–cell interactions (organoid formation) [7,8]; (ii) functionality (growth factor-immobilized/controlled release material) [9]; (iii) optimized microenvironment around cells (ECM- derived hydrogel cultures) [7]; (iv) scaffold (supplying space for tissue-like structure formation); and (v) capillary network of blood vessels (tissue-like structure for mass transfer) [3]. Therefore, the development of hepatocyte-embedded hydrogel-filled scaffold culture technology and the construction of a sufficient capillary network are important for this realization. In recent years, ECM-derived components have been enzy- matically cross-linked and used as culture substrata. Primary hepatocytes were cultured in highly open porous gelatin scaf- folds by microbial transglutaminase (mTGase) cross-linking. The 1369-703X/$ – see front matter. Crown Copyright © 2010 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.bej.2010.09.003