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Protocol no. 7
THE ISOLATION AND CULTURE OF RAT HEPATIC CELLS

This method enables the isolation of different populations of liver cells and describes their subsequent culture.

CONTACT

Cell Culture Group
MRC Toxicology Unit Medical Research Council Laboratories
Woodmansterne Road Carshalton, Surrey SM5 4EF UK
Tel: England - 643 8000

BASIC PROCEDURE

The liver of a rat is cannulated and perfused in situ with buffer, following which it is excised and perfused in a closed system with a collagenase solution. After a period of time the liver begins to break up, at which point it is transferred to a measuring cylinder and culture medium is added. It is then gently agitated to cause the release of cells which are subsequently filtered and allowed to settle out. The parenchymal and non-parenchymal cells form two distinct layers which can be separated.

CRITICAL ASSESSMENT

Cell Isolation A relatively fast, reproducible, procedure to obtain liver cells compared to certain other techniques, e.g. slicing followed by enzymatic/mechanical dispersion. This system also enables isolation of specific cell types without resorting to the gradient centrifugation procedure employed in other methods to separate parenchymal and non-parenchymal cells. Non-parenchymal cells can be further fractionated into Kupffer and endothelial cells by the elutration procedure of Knook et al. (1977). Toxicity testing This isolation procedure enables in vitro toxicity tests to be carried out on both major populations of liver cells i.e. parenchymal and sinusoidal cells.

REFERENCES

  1. Seglen, P.O. (1973) Preparation of rat liver cells. Exp. Cell Res., 82, 391-398 (Modified by using Sigma type I or Boehringer collagenase to isolate non-parenchymal and parenchymal cells, respectively.)
  2. Knook, D.L., Blansjaar, N. & Sleyster, E.Ch. (1977) Isolation and characterisation of Kupffer and endothelial cells from the rat liver. Exp. Cell. Res., 109, 317-329

IP-7 © June 1989