CONSTRUCTION OF LARGE BLOCK OF ENGINEERED LIVER TISSUE SEEDED WITH CO-CULTURED CELLS AND IN VIVO IMPLANTATION RESEARCH_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

QUXiaoli ¹ , WANGMin ² , HEJiankang ¹ ,  LIUYaxiong ¹ ,  ZHOUXinmin ² , LIDichen ¹ , JINZhongmin ^1,3

1. State Key Laboratory for Manufacturing Systems Engineering, Xi'an Jiaotong University, Xi'an Shaanxi, 710049, P. R. China;
2. the 8th Department, Xijing Hospital of Digestive Diseases, the Fourth Military Medical University;
3. Institute of Medical and Biological Engineering, Leeds University, UK;

Corresponding author：

LIUYaxiong, Email: yaxiongliu@mail.xjtu.edu.cn; ZHOUXinmin, Email: zhouxm128@fmmu.edu.cn

Keywords：

Tissue engineered liver; Three-dimensional printing technique; Collagen hydrogel scaffold; Fibroblasts; Primary hepatocytes; Co-culture; Implantation in vivo; Rat

DOI：

10.7507/1002-1892.20140073

Video：

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Objective To construct large block of engineered liver tissue by co-culture of fibroblasts and hepatocytes on collagen hydrogels in vitro and do in vivo implantation research. Methods Silastic mould was prepared using three-dimensional printing technology. The collagen hydrogel scaffold was prepared by collagen hydrogel gel in the silicone mould and was removed. Sprague Dawley rat lung fibroblasts were co-cultured with primary hepatocytes at a ratio of 0.4:1 on the collagen hydrogel scaffold to construct large block of engineered liver tissue in vitro (group B), and primary hepatocytes cultured on the collagen hydrogel scaffold served as control group (group A). The cell morphology was observed, and the liver function was tested at 1, 3, 7, 14, and 21 days after culture. The rat model (n=24) of hepatic cirrhosis was made by subcutaneous injection of carbon tetrachloride. And in vivo implantation study was carried in cirrhosis rat model. The phenotypic characteristics and functional expression of hepatocytes were evaluated at 3, 7, 14, 21, and 28 days after implantation. Results In vitro results indicated that hepatocytes in group B exhibited compact polyhedral cells with round nuclei and high expression of liver function. Moreover, cells aggregated to the most at 7 days. Album production and urea synthesis incresed significantly when compared with group A (P<0.05). In vivo results showed hepatocytes in group B survived for 28 days, and albumin production and urea synthesis were significantly increased. In addition, hepatocytes showed an aggregated distribution and cord-like structures, which was similar to normal liver tissue. Conclusion The large block of engineered liver tissue constructed by co-cultured cells can form tissue similar to normal liver tissue in vivo, and survive for a long time, laying foundations for building more complete engineered liver tissue in the future.

Citation： QUXiaoli, WANGMin, HEJiankang, LIUYaxiong, ZHOUXinmin, LIDichen, JINZhongmin. CONSTRUCTION OF LARGE BLOCK OF ENGINEERED LIVER TISSUE SEEDED WITH CO-CULTURED CELLS AND IN VIVO IMPLANTATION RESEARCH. Chinese Journal of Reparative and Reconstructive Surgery, 2014, 28(3): 325-330. doi: 10.7507/1002-1892.20140073 Copy

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1. Kisseleva T, Gigante E, Brenner DA. Recent advances in liver stem cell therapy. Curr Opin Gastroenterol, 2010, 26(4):395-402.
2. Goulet F, Normand C, Morin O. Cellualr interactions promote tissue-specific function, biomatrix deposition and junctional communication of primary cultured hepatocytes. Hepatology, 1988, 8(5):1010-1018.
3. Gotoh Y, Ishizuka Y, Matsuura T, et al. Spheroid formation and expression of liver-specific functions of human hepatocellular carcinoma-derived FLC-4cells cultured in lactose-silk fibroin conjugate sponges. Biomacromolecules, 2011, 12(5):1532-1539.
4. Ijima H, Kubo T, Hou YT. Primary rat hepatocytes form spheroids on hepatocyte growth factor/heparin-immobilized collagen film and maintain high albumin production. Biochemical Engineering Journal, 2009, 46(2):227-233.
5. Ijima H, Kakeya Y, Yokonuma T, et al. Composition of culture medium is more important than co-culture with hepatic non-parenchymal cells in albumin production activity of primary rat hepatocytes, and the effect was enhanced by hepatocytes spheroid culture in collagen gel. Biochemical Engineering Journal, 2009, 45(3):226-231.
6. Ijima H, Matsuo T, Kawakami K. The mixed coculture effect of primary rat hepatocytes and bone marrow cells is caused by soluble factors derived from bone marrow cells. J Biosci Bioeng, 2008, 105(3):226-231.
7. Riccalton-Banks L, Liew C, Bhandari R, et al. Long-term culture of functional liver tissue:three-dimensional coculture of primary hepatocytes and stellate cells. Tissue Eng, 2003, 9(3):401-410.
8. Sukmana I, Vermette P. The effects of co-culture with fibroblasts and angiogenic growth factors on microvascular maturation and multi-cellular lumen formation in HUVEC-oriented polymer fibre constructs. Biomaterials, 2010, 31(19):5091-5099.
9. Rajan N, Habermehl J, Coté MF, et al. Preparation of ready-to-use, storable and reconstituted type I collagen from rat tail tendon for tissue engineering applications. Nat Protoc, 2006, 1(6):2753-2758.
10. Chen AA, Thomas DK, Ong LL, et al. Humanized mice with ectopic artificial liver tissues. Proc Natl Acad Sci U S A, 2011, 108(29):11842-11847.
11. Nishikawa M, Kojima N, Komori K, et al. Enhanced maintenance and functions of rat hepatocytes induced by combination of on-site oxygenation and coculture with fibroblasts. J Biotechnol, 2008, 133(2):253-260.
12. Sakai Y, Koike M, Hasegawa H, et al. Rapid fabricating technique for multi-layered human hepatic cell sheets by forceful contraction of the fibroblast monolayer. PLoS One, 2013, 8(7):e70970.
13. Kim K, Ohashi K, Utoh R, et al. Preserved liver-specific functions of hepatocytes in 3D co-culture with endothelial cell sheets. Biomaterials, 2012, 33(5):1406-1413.

Chinese Journal of Reparative and Reconstructive Surgery

CONSTRUCTION OF LARGE BLOCK OF ENGINEERED LIVER TISSUE SEEDED WITH CO-CULTURED CELLS AND IN VIVO IMPLANTATION RESEARCH

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