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http://dx.doi.org/10.1080/19768354.2011.555140

A cost-effective and simple culture method for primary hepatocytes  

Adaya, Sezin (Bioengineering Department, Engineering Faculty, Ege University)
Hasircib, Nesrin (Department of Chemistry, Faculty of Arts and Sciences, Middle East Technical University)
Gurhana, Ismet Deliloglu (Bioengineering Department, Engineering Faculty, Ege University)
Publication Information
Animal cells and systems / v.15, no.1, 2011 , pp. 19-27 More about this Journal
Abstract
Hepatocytes, the major epithelial cells of the liver, maintain their morphology in culture dishes coated with extracellular matrix (ECM) components such as collagen and fibronectin or biodegradable polymers (e.g. chitosan, gelatin). In these coated dishes, survival of cells and maintaining of liver-specific functions may increase. The aim of this study was to determine a suitable, cost-effective and simple system for hepatocyte isolation and culture which may be useful for various applications such as in vitro toxicology studies, hepatocyte transplantation and bioartificial liver (BAL) systems. In order to obtain primary cultures, hepatocytes were isolated from liver by an enzymatic method and cultured on plates coated with collagen, chitosan or gelatin. Collagen, gelatin-sandwich and gelatin-cell mixture methods were also evaluated. Morphology and attachment of the cells were observed by inverted microscope and scanning electron microscope (SEM). An MTT assay was used to determine cell viability and mitochondrial activity.
Keywords
collagen; chitosan; gelatin; enzymatic isolation; primary hepatocytes;
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1 Suh JKF, Matthew HWT. 2000. Application of chitosan-based polysaccharide biomaterials in cartilage tissue engineering: a review. Biomaterials. 21:2589-2598.   DOI   ScienceOn
2 Tokiwa T, Kano J, Kodama M, Matsumura T. 1997. Multilayer rat hepatocyte aggregates formed on expanded polytetrafluoroethylene surface. Cytotechnology. 25: 137-144.   DOI   ScienceOn
3 Li K, Wang Y, Miao Z, Xu D, Tang Y, Feng M. 2004. Chitosan/gelatin composite microcarrier for hepatocyte culture. Biotechnol Lett. 26:879-883.   DOI
4 Mao JS, Zhao LG, Yin YJ, Yao KD. 2003. Structure and properties of bilayer chitosan-gelatin scaffolds. Biomaterials. 24:1067-1074.   DOI   ScienceOn
5 Mao JS, Cui YL, Wang XH, Sun Y, Yin YJ, Zhao HM, Yao K. 2004. A preliminary study on chitosan and gelatin polyelectrolyte complex cytocompatibility by cell cycle and apoptosis analysis. Biomaterials. 25:3973-3981.   DOI   ScienceOn
6 Moscato S, Mattii L, D'Alessandro D, Cascone MG, Lazzeri L, Serino LP, Dolfi A, Bernardini N. 2008. Interaction of human gingival fibroblasts with PVA/gelatine sponges. Micron. 39:569-579.   DOI   ScienceOn
7 Pachence JM. 1996. Collagen-based devices for soft tissue repair. J Biomed Mater Res B. 33:35-40.   DOI
8 Huang Y, Onyeri S, Siewe M, Moshfeghian A, Madihally SV. 2005. In vitro characterization of chitosan-gelatin scaffolds for tissue engineering. Biomaterials. 26:7616-7627.   DOI   ScienceOn
9 Kim BS, Baez CE, Atala A. 2000. Biomaterials for tissue engineering. World J Urol. 18:2-9.   DOI   ScienceOn
10 Kravchenko L, Petrenko A, Shanina I, Fuller B. 2002. A simple non-enzymatic method for the isolation of functional rat hepatocytes. Cell Biol Int. 26: 1003-1006.   DOI   ScienceOn
11 Lee JE, Park JC, Hwang YS, Kim JK, Kim JG, Suh H. 2001. Characterization of UV-irradiated dense/porous collagen membranes: morphology, enzymatic degradation, and mechanical properties. Yonsei Med J. 42: 172-179.   DOI
12 Glicklis R, Shapiro L, Agbaria R, Merchuk JC. 2000. Hepatocyte behavior within three-dimensional porous alginate scaffolds. Biotechnol Bioeng. 67: 345-353.
13 Li K, Qu X, Wang Y, Tang Y, Qin D, Wang Y, Feng M. 2005. Improved performance of primary rat hepatocytes on blended natural polymers. J Biomed Mater Res A. 75:268-274.
14 Eschbach E, Chatterjee SS, Noldner M, Gottwald E, Dertinger H, Weibezahn KL, Knedlitschek G. 2005. Microstructured scaffolds for liver tissue cultures of high cell density: morphological and biochemical characterization of tissue aggregates. J Cell Biochem. 95:243-255.   DOI   ScienceOn
15 Fukuda J, Sakai Y, Nakazawa K. 2006. Novel hepatocyte culture system developed using micro fabrication and collagen/polyethylene glycol microcontact printing. Biomaterials. 27:1061-1070.   DOI   ScienceOn
16 Battle T, Stacey G. 2001. Cell culture models for hepatotoxicology. Cell Biol Toxicol. 17:287-289.   DOI   ScienceOn
17 Guguen-Guillouzo C. 2002. Isolation and culture of animal and human hepatocytes. In: Freshney RI, Freshney MG, editors. Culture of epithelial cells. New York (NY): Wiley-Liss. p. 337-379.
18 Hirano S, Itakura C, Seino H, Akiyama Y, Nonaka I, Kanbara N, Kawakami T. 1990. Chitosan as an ingredient for domestic-animal feeds. J Agric Food Chem. 38:1214-1217.   DOI
19 Amaral IF, Lamghari M, Sousa SR, Sampaio P, Barbosa M. 2005. Rat bone marrow stromal cell osteogenic differentiation and fibronectin adsorption on chitosan membranes: The effect of the degree of acetylation. J Biomed Mater Res A. 75A:387-397.   DOI   ScienceOn
20 Chatelet C, Damour O, Domard A. 2001. Influence of the degree of acetylation on some biological properties of chitosan films. Biomaterials. 22:261-268.   DOI   ScienceOn
21 Schmelzer E, Zhang L, Bruce A, Wauthier E, Ludlow J, Yao H, Moss N, Melhem A, McClelland R, Turner W, et al. 2007. Human hepatic stem cells from fetal and postnatal donors. J Exp Med. 204:1973-1987.   DOI   ScienceOn
22 Chupa JM, Foster AM, Sumner SR, Madihally SV, Matthew HW 2000. Vascular cell responses to polysaccharide materials: in vitro and in vivo evaluations. Biomaterials. 21:2315-2322.   DOI   ScienceOn
23 Dunn JCY, Yarmush ML, Koebe HG, Tompkins RG. 1989. Hepatocyte function and extracellular matrix geometry: long-term culture in a sandwich configuration. FASEB J. 3:174-177.   DOI
24 Aiedeh K, Gianasi E, Orienti I, Zecchi V. 1997. Chitosan microcapsules as controlled release systems for insulin. J Microencapsul. 14:567-576.   DOI   ScienceOn
25 Allen JW, Hassanein T, Bhatia S. 2001. Advances in bioartificial liver devices. Hepatology. 34:447-455.
26 Wang XH, Li DP, Wang WJ, Feng QL, Cui FZ, Xu YX, Song XH, van der Werf M. 2003. Crosslinked collagen/chitosan matrix for artificial livers. Biomaterials. 24:3213-3220.   DOI   ScienceOn
27 Zavan B, Brun P, Vindigni V, Amadori A, Habeler W, Pontisso P, Montemurro D, Abatangelo G, Cortivo R. 2005. Extracellular matrix-enrich ted polymeric scaffolds as a substrate for hepatocyte cultures: in vitro and in vivo studies. Biomaterials. 26:7038-7045.   DOI   ScienceOn
28 Zhu A, Zhang M, Wu J, Shen J. 2002. Covalent immobilization of chitosan/heparin complex with a photosensitive hetero-bifunctional crosslinking reagent on PLA surface. Biomaterials. 23:4657-4665.   DOI   ScienceOn
29 Park KH, Song SC. 2006. Morphology of spheroidal hepatocytes within injectable, biodegradable, and thermosensitive poly(organophosphazene) hydrogel as cell delivery vehicle. J Biosci Bioeng. 101:238-242.   DOI   ScienceOn
30 Puviani AC, Ottolenghi C, Tassinari B, Pazzi P, Morsiani E. 1998. An update on high-yield hepatocyte isolation methods and on the potential clinical use of isolated liver cells. Comp Biochem Physiol A. 121:99-109.
31 Shen C, Zhang G, Qiu H, Meng Q. 2006. Acetaminophen-induced hepatotoxicity of gel entrapped rat hepatocytes in hollow fibers. Chem Biol Interact. 162:53-61.   DOI   ScienceOn