Abstract
Mimicking the in vivo microenvironment is one of the current strategies to maintain liver-specific functionality in primary cultured hepatocytes for long periods. Freshly isolated hepatocytes entrapped in collagen gel type I (collagen gel immobilization culture) or sandwiched between two layers of hydrated collagen type I (collagen gel sandwich culture) are known to display liver-specific functions (e.g., biotransformation capacity) for more than 6 wk. We describe how to set up both types of organotypical hepatocyte culture systems. Besides a detailed protocol, we give some practical tips, taken from our own experience with long-term hepatocyte culture.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
LeCluyse, E. L., Bullock, P. L., and Parkinson, A. (1996) Strategies for restoration and maintenance of normal hepatic structure and function in long-term cultures of rat hepatocytes. Adv. Drug Deliv. Rev. 22, 133–186.
Maher, J. J., and Bissell, D. M. (1993) Cell-matrix interactions in liver. Semin. Cell Biol. 4, 189–201.
Hasirci, V., Berthiaume, F., Bondre, S. P., et al. (2001) Expression of liver-specific functions by rat hepatocytes seeded in treated poly(lactic-co-glycolic) acid biodegradable foams. Tissue Eng. 7, 385–394.
Yang, T. H., Miyoshi, H., and Ohshima, N. (2001) Novel cell immobilization method utilizing centrifugal force to achieve high-density hepatocyte culture in porous scaffold. J. Biomed. Mater. Res. 55, 379–386.
Pahernik, S. A., Thasler, W. E., Doser, M., et al. (2001) High density culturing of porcine hepatocytes immobilized on nonwoven polyurethane-based biomatrices. Cells Tissues Organs 168, 170–177.
Linti, C., Zipfel, A., Schenk, M., et al. (2002) Cultivation of porcine hepatocytes in polyurethane nonwovens as part of a biohybrid liver support system. Int. J. Artif. Organs 25, 994–1000.
Hanada, S., Kayano, H., Jiang, J., et al. (2003) Enhanced in vitro maturation of subcultivated fetal human hepatocytes in three dimensional culture using poly-L-lactic acid scaffolds in the presence of oncostatin M. Int. J. Artif. Organs 26, 943–951.
Richert, L., Binda, D., Hamilton, G., et al. (2002) Evaluation of the effect of culture configuration on morphology, survival time, antioxidant status and metabolic capacities of cultured rat hepatocytes. Toxicol. In Vitro 16, 89–99.
LeCluyse, E. L. (2001) Human hepatocyte culture systems for the in vitro evaluation of cytochrome P450 expression and regulation. Eur. J. Pharm. Sci. 13, 343–368.
Gómez-Lechón, M. J., Jover, R., Donato, T., et al. (1998) Long-term expression of differentiated functions in hepatocytes cultured in three-dimensional collagen matrix. J. Cell. Physiol. 177, 553–562.
Gómez-Lechón, M. J., Jover, R., Donato, T., Ponsoda, X., and Castell, J. V. (2000) Expression of liver specific-genes in hepatocytes cultured in collagen gel matrix. Prog. Mol. Subcell. Biol. 25, 89–104.
Beken, S., Pauwels, M., Pahernik, S., Koebe, H.-G., Vercruysse, A., and Rogiers, V. (1997) Collagen gel sandwich and immobilization cultures of rat hepatocytes: problems encountered in expressing glutathione S-transferase activities. Toxicol. In Vitro 11, 741–752.
Beken, S., Pahernik, S., Koebe, H.-G., Vercruysse, A., and Rogiers, V. (1997) Cell morphology, albumin secretion, and glutathione S-transferase expression in collagen gel sandwich and immobilization cultures of rat hepatocytes. Toxicol. In Vitro 11, 409–416.
Dunn, J. C., Tompkins, R. G., and Yarmush, M. L. (1991) Long-term in vitro function of adult hepatocytes in a collagen sandwich configuration. Biotechnol. Prog. 7, 237–245.
Berthiaume, F., Moghe, P. V., Toner, M., and Yarmush, M. L. (1996) Effect of extracellular matrix topology on cell structure, function, and physiological responsiveness: hepatocytes cultured in a sandwich configuration. FASEB J. 10, 1471–1484.
De Smet, K., Cavin, C., Vercruysse, A., and Rogiers, V. (2001) Collagen type I gel cultures of adult rat hepatocytes as a screening induction model for cytochrome P450-dependent enzymes. ATLA 29, 179–192.
Beken, S., Depreter, M., Roels, F., et al. (2005) The use of collagen gel cultures of rat hepatocytes as screening tools for inducers of glutathione S-transferase isoenzymes. ATLA, in press.
Koebe, H.-G., Pahernik, S., Eyer, P., and Schildberg, F.-W. (1994) Collagen gel immobilization: a useful cell culture technique for long-term metabolic studies in human hepatocytes. Xenobiotica 24, 95–107.
Elsdale, T., and Bard, J. (1972) Collagen substrata for studies on cell behavior. J. Cell Biol. 54, 626–637.
Beken, S., De Smet, K., Depreter, M., Roels, F., Vercruysse, A., and Rogiers, V. (2001) Effects of L-proline on phase I and phase II xenobiotic biotransformation capacities of rat and human hepatocytes in long-term collagen gel cultures. ATLA 29, 35–53.
De Smet, K., Bruning, T., Blaszkewicz, M., Bolt, H. M., Vercruysse, A., and Rogiers, V. (2000) Biotransformation of trichloroethylene in collagen gel sandwich cultures of rat hepatocytes. Arch. Toxicol. 74, 587–592.
De Smet, K., Loyer, P., Gilot, D., Vercruysse, A., Rogiers, V., and Guguen-Guillouzo, C. (2001) Effects of epidermal growth factor on CYP inducibility by xenobiotics, DNA replication, and caspase activations in collagen I gel sandwich cultures of rat hepatocytes. Biochem. Pharmacol. 61, 1293–1303.
Unger, J. K., Kuehlein, G., Schroers, A., Gerlach, J. C., and Rossaint, R. (2001) Adsorption of xenobiotics to plastic tubing incorporated into dynamic in vitro systems used in pharmacological research: limits and progress. Biomaterials 22, 2031–2037.
Acknowledgments
This work was supported by grants from the Fund of Scientific Research Flanders (FWO), Belgium; the Research Council of the Vrije Universiteit Brussel, Belgium; and the EU 6th Framework Program.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2006 Humana Press Inc.
About this protocol
Cite this protocol
Vinken, M. et al. (2006). Rat Hepatocyte Cultures. In: Phillips, I.R., Shephard, E.A. (eds) Cytochrome P450 Protocols. Methods in Molecular Biology, vol 320. Humana Press, Totowa, NJ. https://doi.org/10.1385/1-59259-998-2:247
Download citation
DOI: https://doi.org/10.1385/1-59259-998-2:247
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-58829-441-8
Online ISBN: 978-1-59259-998-1
eBook Packages: Springer Protocols