Development of an intact hepatocyte activation system for routine use with the mouse lymphoma assay

We have developed a method for cocultivating primary rat hepatocytes with L5178Y/TK+/- -3.7.2C mouse lymphoma cells. This method should provide a means of stimulating more closely in-vivo metabolism compared to metabolism by liver homogenates, while still being useful for routine screening. Hepatocytes were isolated from 200-250 gm adult male Sprague-Dawley rats; 1 X 10(6) viable hepatocytes were seeded per flask. Rapid attachment of the hepatocytes (2 hr) was obtained by using fibronectin-coated 25-cm2 tissue culture flasks. Cocultivated cultures were incubated at 37 degrees C on a platform rocker at 32 oscillations per minute. A 16-hr cocultivated period was selected. With this hepatocyte activation methodology, CP, DMN, DMBA, and B(a)P, genotoxins that require metabolic activation, could be detected as mutagens in L5178Y/TK+/- cells.