THE MORPHOLOGY AND FUNCTION OF RABBIT HEPATOCYTES ISOLATED USING ETHYLENEDIAMINETETRAACETATE1

Abstract

The enzyme digestion technique using collagenase is used in most studies that describe hepatocyte isolation, including those in which hepatocytes are isolated for transplantation. The use of collagenase, however, has several drawbacks. We describe the use of a highly concentrated ethylenediaminetetraacetate solution for isolation of hepatocytes followed by purification using a Percoll gradient in the rabbit model. Isolated hepatocytes were then preserved at 4 degrees C for up to three days in either University of Wisconsin solution or Dulbecco's modified eagle's medium. Morphological studies of hepatocytes at 24 hr and 72 hr in either medium were performed using Papanicolaou and PAS stains of cytopreparations for the presence of glycogen. Similarly, functional studies of the preserved hepatocytes included LDH release, total tissue water content, and amount of 99m-technetium mebrofenin uptake. The use of EDTA perfusion for hepatocyte isolation was highly reproducible in this model. The cell yield was comparable to that achieved previously using collagenase. The morphologic studies demonstrated pure hepatocytes with well-preserved architecture when preserved for up to three days in UW solution. Functional studies showed a statistically significant lower LDH release and total tissue water content and a higher technetium-99m mebrofenin uptake for hepatocytes preserved in UW solution. We conclude that a pure and viable hepatocyte suspension can be obtained from rabbit livers using concentrated EDTA solutions. These hepatocytes can be well preserved at 4 degrees C for up to 72 hr in UW solution, based on morphologic and functional criteria.