Effects of various medium formulations and attachment substrata on the performance of cultured ruminant hepatocytes in biotransformation studies

Abstract

1. A procedure for the isolation and primary culture of hepatocytes from goat and cattle is described. Hepatocyte culture performance was monitored for 51 h by measuring viability, cytochrome P-450 maintenance, dealkylation of scoparone and ethylmorphine, and glucuronidation of phenol red. 2. Culture medium composition is discussed in relation to differences between splanchnic blood composition of ruminant and monogastric animal species. Main differences are in glucose and volatile fatty acid concentrations. Modified Williams' E culture medium did not yield higher culture performance than non-modified Williams' E. 3. Coating of culture dishes with either collagen or fibronectin did not improve culture performance. 4. Williams' E, although developed for rodent cells, proves to be a suitable basal medium for ruminant hepatocytes. In this medium, culture quality is high for at least several days. 5. In cultured goat hepatocytes, biotransformation rate for scoparone amounted to 20 nmol/mg protein per h, for ethylmorphine 96 nmol/mg protein per h and for phenol red 2 nmol/mg protein per h. Biotransformation activity in cow hepatocytes is approximately half that in goat hepatocytes.

Preparation of extended in vitro cultures of bovine hepatocytes that are hormonally responsive2

“…Cells isolated from goats have been used in both hepatocyte suspension (Aiello and Armentano, 1987a,b;Aiello et al, 1989) and monolayer culture systems (Van Klooster et al, 1992). However, in the present study, satisfactory monolayers were obtained with much lower repeatability from goat liver.…”

“…Several abstracts (Donkin et al, 1990(Donkin et al, , 1991(Donkin et al, , 1992Cadorniga et al, 1992) and one paper (Emmison et al, 1991) have reported the use of ruminant parenchymal cell monolayers t o study hormonal regulation of hepatic metabolism. Xenobiotic metabolism has also been investigated using ruminant hepatocyte monolayers (Shull et al, 1986;Van Klooster et al, 1992). The purpose of this report is to provide a complete description of the technique for establishing bovine hepatocyte monolayers and to characterize changes in cellular metabolism during their extended culture.…”

Xenobiotic Metabolism by Cultured Primary Porcine Hepatocytes

“…These attempts include modifying the composition of the medium (by addition of hormones, chemicals, ligands, and/or inducers to the culture medium) or by incorporating different cell types as in a co-culture model. [34][35][36][37] From their work, it appears that the life time of the primary cultures can be extended by modifying the conditions of the culture. Perhaps one of the significant findings in our study is that porcine hepatocyte culture supports the maintenance of cyp2C19 and 3A4, important human iosforms that are involved in the metabolism of many xenobiotics such as diazepam, as well as phase II glucuronidation activities without a need for induction.…”

In vitro metabolism of cyadox in rat, chicken and swine using ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry

“…Liver microsomes is one of the most commonly used in vitro systems because it is cheaper, has good reproducibility and has simple preparation by using differential centrifugation [14]. In addition, the primary hepatocytes culture model played a significant role in the research of phase I and phase II drug biotransformations [15,16], which provided the most in vivo-like responses [17]. Gastrointestinal microflora was also necessary for the metabolism in vitro owing to various toxicological sequelae by intestinal microflora [18,19].…”