Culture of bovine hepatocytes: a non-perfusion technique for cell isolation
- 253 Downloads
In this work we have studied the isolation and culture of mature bovine hepatocytes on plastic dishes without exogenous matrix. The liver has been disaggregated in a collagenase solution instead of undergoing a perfusion step. After a few days in culture, the plates showed several clusters of different cell types. Although the average yield was 1.60±0.57×108 viable liver cells per gram of tissue, these cultures were formed by non-parenchymal cells and only very few or none by parenchymal cells. In these cultures, actin structures used as a marker for Stellate (Ito) cells have been visualized by immunocytochemical techniques. In order to increase the proportion of parenchymal cells a centrifugation on Percoll, which separates cell sub-populations, has been introduced. Though the yield was lower than in the previous method, these pre-purified cultures were only composed of hepatocytes. It has been shown that these cells exhibited albumin synthesis, which is a specific hepatocytes function. In addition, these cultures were capable of producing metabolites of 7-ethoxycoumarin at a higher rate than non purified cell cultures. Therefore this simplified procedure for the isolation and culture of functional and viable hepatocytes may be applied for in vitro studies in bovine.
KeywordsBovine hepatocytes Cell culture Cell isolation Non-perfusion Purification of parenchymal cells Bovine liver
bovine serum albumin
foetal bovine serum
phosphate buffered saline
3,3′ diaminobenzidinetetrachloride chromogenic-substrate system
Unable to display preview. Download preview PDF.
This work was supported by International Foundation for Science, Grant E-3062. The authors thank to EcoCarnes S.A., Argentina, for providing bovine livers.
- Alston-Smith J, Pertoft H (1995) Isolation of liver cells: a symtem for obtaining pure cells in monolayer cultures from a single rat liver. In: Doyle A, Griffiths JB, Newell DG (eds) Cell and tissue culture. Laboratory Procedures, John Wiley & Sons, New York, USA, pp 12B: 14.1–14.9Google Scholar
- Berry MN, Edwards AM, Barritt GJ (1991) Isolated hepatocytes preparation, properties and applications. In: Burdon RH, van Knippenbergr PH (eds) Laboratory techniques in biochemistry and molecular biology, Elsevier Science Publishers, Amsterdam, The NetherlandsGoogle Scholar
- Donkin SS, Armentano LE (1993) Preparation of extended in vitro cultures of bovine hepatocytes that are hormonally responsive. J Anim Sci 71:2218–2227Google Scholar
- Forsell JH, Jesse BW, Shull LR (1985) A technique for isolation of bovine hepatocytes. J Anim Sci 60:1597–1609Google Scholar
- Fry JF, Jones CA, Wiebkin P, Bellemann P, Bridges JW (1976) The enzymic isolation of adult rat hepatocytes in a functional and viable state. Anal Chem 71:341–350Google Scholar
- Funaki N, Tanaka J, Sugiyama T, Ohshio G, Nonaka A, Yotsumoto F, Sugie T, Imamura M (2002) Successive cultures of mature hepatocyte autotransplantation to assist liver function after liver resection for cancer. Oncol Reports 9:713–721Google Scholar
- Gibson-D'Ambrosio RE, D'Ambrosio SM (1996) Replicative and Functional Cultures of Normal Human, Hepatocytes. In: Doyle A, Griffiths JB, Newell DG, (eds) Cell & Tissue Culture. Laboratory Procedures, John Wiley & Sons, New York, USA, pp 12B: 17.1–17.16Google Scholar
- Nakamura T, Tomita Y, Ichihara A (1983a) Density-dependent growth control of adult rat hepatocytes in primary culture. J Biochem 94:1029–1035Google Scholar
- Shull L, Kirsch DG, Lohse CL, Carlson GP, Doody LA, Wisniewski JA (1986) Xenobiotic metabolism in suspension and primary cultures of isolated hepatocytes prepared from caudate process of bovine liver. Am J Vet Res 49(9):2043–2052Google Scholar