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A Technique for Isolation of Bovine Hepatocytes¹

Michigan State University, East Lansing 48824 and University of California, Davis 95616

Abstract

A technique for preparing viable and functional isolated hepatocytes from cattle liver is described. The basic procedure, which was adapted from published methods established for laboratory species, employed a two-step in vitro vascular perfusion of the caudate lobe: (1) perfusion with a calcium-free buffer containing ethylene bis(oxyethylenenitrilo)tetraacetic acid (EGTA) for removal of blood cells and extra-cellular calcium and (2) perfusion with calcium-fortified buffer containing collagenase for cell dissociation. Hepatocyte suspensions prepared from the caudate lobes of 20 cattle possessed a mean viability of 81.3% as determined by trypan blue exclusion. Mean yield was 2.2 x 10⁷ viable hepatocytes/g of liver (wet wt). Viable hepatocytes utilized O₂ at a rate 2.82 times greater than nonviable hepatocytes. Biochemical function of the hepatocyte suspensions was assessed by rates of gluconeogenesis and fatty acid oxidation. Glucose production from added lactate ranged from .88 to 1.47 µmol·min^–1·g^–1 of liver tissue (dry wt). Both gluconeogenic and fatty acid oxidation rates were substantially greater in isolated hepatocytes when compared with liver slices. Isolated hepatocyte contained .398 ± .033 (SE) nmol cytochromes P-450/mg microsomal protein and .285 ± .025 nmol cytochrome b₅/mg microsomal protein, which was comparable with amounts in liver tissue from the same animals (.568 ± .056 and .298 ± .033 nmol/mg protein, respectively). No significant decline of either cytochrome was detectable for isolated hepatocytes for up to 5.5 h after euthanasia. The potential usefulness of isolated bovine hepatocytes in xenobiotic metabolism studies is illustrated by the epoxidation of aldrin.

¹ This research was supported in part by a grant (1 R23FD01 224-01) from The Center for Veterinary Medicine, Food and Drug Administration, Rockville, MD. Michigan Agr. Exp. Sta. Journal Article No. 11389.

² Dept. of Anim. Sci., Center for Environ. Toxicol. Present address: Dept. of Food Sci. and Human Nutr.

³ Dept. of Biochem., School of Medicine, Case Western Reserve Univ. Cleveland, OH 44106.

⁴ Dept. of Environ. Toxicol., Univ. of California, Davis 95616.

⁵ Appreciation is expressed by the authors to Dr. John R. Kateley for his comments and to Dr. Edgardo Cardozo for his assistance in the statistical evaluation of the data.