A Technique for Isolation of Bovine Hepatocytes

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 extracellular 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 10(7) 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 mumol X min-1 X 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 bs/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.

Evaluation of Tissue Preparation forin vitroStudy of Hepatic Long-Chain Fatty Acid Metabolism

“…When comparing the results from the present experiment with results from the literature where live ruminants have been biopsied (Aiello et al, 1984;Forsell et al, 1985;Jesse et al, 1986a,b;Drackley et al, 1991c;Grum et al, 1996b), it seems that the production of CO 2 and ASP in the present experiment is twice as low or even lower, but with a similar good coef® cient of variation. The two most distinct factors explaining the lower production rate in this experiment are the medium used and the tissue sampling procedure.…”

“…ASP were analysed as described by Grum et al (1994). ASP have been evaluated to be mainly ( \ 70%) b-hydroxybutyrat e and acetoacetate (Forsell et al, 1985) and, therefore, an estimation of ketogenesis. For a further de® nition, see Veerkamp et al (1986).…”

“…However, major differences in liver tissue metabolism values obtained with in ×itro techniques may be due to differences in preparing the tissue. Traditionally, liver slices have been prepared either by hand-cutting with razor blades (Leng & Annison, 1963;Beylot, 1996), using a Stadie ± Riggs hand microtome (SR) (Aiello et al, 1984;Forsell et al, 1985;Mills et al, 1986;Drackley et al, 1991c;Veenhuizen et al, 1991;Knapp et al, 1992), or using a motorized precisioncut tissue slicer such as the Krumdieck tissue slicer (Drackley et al, 1991a,b;Grum et al, 1996a,b). The above-mentioned methods require relatively large tissue samples, from 3 to 7 g of tissue (Drackley et al, *Corresponding author.…”

Comparison of Pig Hepatocyte Isolation Using Intraoperative Perfusion without Warm Ischemia and Isolation of Cells from Abattoir Organs after Warm Ischemia

“…The intraoperative perfusion technique was supe-Artij'Orgnns, Vol. 17, No, 11,1993 rior with respect to yield and viability of the cells. Recently, we have investigated the relationship of animal weight and hepatocyte isolation, and we found an increase of yield and viability using organs from animals with a mass less than 25 kg.…”

Culture of bovine hepatocytes: a non-perfusion technique for cell isolation

“…Usually a perfusion step is an integral part of cell disaggregation method (Forsell et al 1985;Shull et al 1986;Donkin and Armentano 1993). However, when freshly isolated samples of liver from large mammals, like bovine, are used, the perfusion step is technologically demanding.…”