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

Abstract

Hepatocytes isolated from male dairy calves were used in monolayer culture or in suspension culture to determine their suitability for the study of hormonal regulation of hepatic gluconeogenesis. The rate of gluconeogenesis (nanomoles of 2.5 mM [2-14C]propionate incorporated into glucose.microgram of DNA-1.hour-1) was higher for monolayers than for suspension cultures. Gluconeogenesis and ureagenesis (nanomoles of urea N formed.microgram of DNA-1.3 hours-1) were similar in monolayers cultured for 24 and 48 h but declined by 120 h. Ureagenesis was barely detectable in suspension cultures. Glucagon (10 nM) increased gluconeogenesis from propionate in monolayers but was without effect on suspension cultures. Actinomycin D (800 nM) and cycloheximide (200 microM) abolished glucagon stimulation of gluconeogenesis, suggesting that glucagon acts to mediate gene expression. Prolonged exposure (45 h) of monolayers to insulin (1,000 nM) decreased basal gluconeogenic rates but did not affect glucagon-stimulated gluconeogenesis. Prior incubation with glucose or valerate did not affect gluconeogenesis. Cells can be successfully maintained in serum-free media for 41 h at the expense of diminished basal gluconeogenic activity. Culture of bovine hepatocytes as monolayers provides a useful tool for the study of chronic and acute hormonal regulation of specific liver functions in the bovine.

Regulation of cellular glutathione peroxidase by different forms and concentrations of selenium in primary cultured bovine hepatocytes

“…Hepatocyte isolation was performed by the collagenase perfusion method as previously described [28,29], with some modifications. The liver lobe was rewarmed by preperfusion for 10-12 min with the same calcium-free HEPES buffer (37-38°C) but without EGTA.…”

Effect of NEFA and Glucose Levels on CPT-I mRNA Expression and Translation in Cultured Bovine Hepatocytes

“…The cells were then cultured at 37C in 5% CO 2 . Every 24 hr, the media were aspirated and refreshed [4,18]. At 72 hr, liver cells was cultured with glucose-and NEFA-free RPMI 1640 culture medium.…”

Isolation and cultivation of adult primary bovine hepatocytes from abattoir derived liver

“…Primary hepatocytes obtained from adult mice and rats are widely used for toxicological and metabolic studies and well standardized (Hewitt et al, 2007[5]; Shulman and Nahmias, 2013[12]). Currently, the majority of research on bovine hepatocytes is done with cells obtained from calves in monolayer culture (Donkin and Armentano, 1993[1]; Zhang et al, 2016[17]). For primary hepatocytes however sandwich culture is currently the most suitable method.…”