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Cytotechnology

, Volume 51, Issue 2, pp 51–56 | Cite as

Culture of bovine hepatocytes: a non-perfusion technique for cell isolation

  • Viviana Graciela Spotorno
  • Alejandra Hidalgo
  • Mariana Barbich
  • Alicia Lorenti
  • Osvaldo Zabal
Article

Abstract

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.

Keywords

Bovine hepatocytes Cell culture Cell isolation Non-perfusion Purification of parenchymal cells Bovine liver 

Abbreviations

BSA

bovine serum albumin

FBS

foetal bovine serum

PBS

phosphate buffered saline

ATB

antibiotics

DAB

3,3′ diaminobenzidinetetrachloride chromogenic-substrate system

7-EC

7-ethoxycoumarin

7-OH

7-hydroxycoumarin

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Notes

Acknowledgements

This work was supported by International Foundation for Science, Grant E-3062. The authors thank to EcoCarnes S.A., Argentina, for providing bovine livers.

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Copyright information

© Springer Science+Business Media B.V. 2006

Authors and Affiliations

  • Viviana Graciela Spotorno
    • 1
  • Alejandra Hidalgo
    • 2
  • Mariana Barbich
    • 2
  • Alicia Lorenti
    • 2
  • Osvaldo Zabal
    • 3
  1. 1.Instituto Tecnología de Alimentos. C.I.A.Instituto Nacional de Tecnología Agropecuaria (INTA)Buenos AiresArgentina
  2. 2.Instituto de Ciencias Básicas y Medicina ExperimentalHospital Italiano de Buenos AiresBuenos AiresArgentina
  3. 3.Instituto de Virología. C.I.C.V.yA.Instituto Nacional de Tecnología Agropecuaria (INTA)Buenos AiresArgentina

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