In Vitro

, Volume 17, Issue 10, pp 913–925

Mouse liver cell culture

I. Hepatocyte isolation
  • James E. Klaunig
  • Peter J. Goldblatt
  • David E. Hinton
  • Michael M. Lipsky
  • Jacob Chacko
  • Benjamin F. Trump
Article

DOI: 10.1007/BF02618288

Cite this article as:
Klaunig, J.E., Goldblatt, P.J., Hinton, D.E. et al. In Vitro (1981) 17: 913. doi:10.1007/BF02618288

Summary

A method for isolation of mouse liver cells by a two-step perfusion with calcium and magnesium-free Hanks' salt solution followed by a medium containing collagenase is described. Several variations of the commonly used procedure for rat liver cell isolation were quantitatively compared with respect to cell yield and viability. The optimal isolation technique involved perfusion through the hepatic portal vein and routinely produced an average of 2.3×106 viable liver cells/g body weight. Optimal perfusate collagenase concentration was found to be 100 U of enzyme activity per milliliter of perfusate.

Light and electron microscopic evaluation of liver morphology after several steps of the isolation showed distinct morphologic changes in hepatocytes and other liver cells during perfusion. After perfusion with Hanks' calcium-and magnesium-free solution, many hepatocytes exhibited early reversible cell injury. These changes included vesiculation and slight swelling of the endoplasmic reticulum as well as mitochondrial matrix condensation. Subsequent to perfusion with collagenase, the majority of hepatocytes appeared connected to one another only by tight junctional complexes at the bile canaliculi. Multiple evaginations were see on the outer membrane resembling microvilli and probably represented the remains of cell-to-cell interdigitations between hepatocytes and sinusoidal lining cells from the space of Disse. The cytoplasmic injury seen after Hanks' perfusion was reversed after collagenase perfusion. After mechanical dispersion, isolated mouse hepatocytes were spherical in shape and existed as individual cells; many (80 to 85%) were binucleated under phase contrast light microscopy. By electron microscopy, cells appeared morphologically similar in cytoplasmic constitution to that seen in intact nonaltered liver cells.

Key words

mouse hepatocyte isolation perfusion morphology 

Copyright information

© Tissue Culture Association, Inc 1981

Authors and Affiliations

  • James E. Klaunig
    • 1
  • Peter J. Goldblatt
    • 1
  • David E. Hinton
    • 3
  • Michael M. Lipsky
    • 2
  • Jacob Chacko
    • 2
  • Benjamin F. Trump
    • 2
  1. 1.Department of PathologyMedical College of OhioToledo
  2. 2.Department of PathologyUniversity of Maryland School of MedicineBaltimore
  3. 3.Department of AnatomyWest Virginia University School of MedicineMorgantown