A Nonperfusion‐Based Method of Hepatic Cell Isolation and Development of Fatty Liver Disease Model for Dairy Cattle

Abstract

Fatty liver disease (FLD) is a major metabolic disorder of over‐conditioned periparturient dairy cattle and can precede many costly comorbidities, such as mastitis and ketosis. While management practices can decrease the incidence rate of FLD, the etiology is not fully understood. In vivo studies using large animals are expensive, difficult to perform, and time‐consuming; so, an in vitro model of FLD would be valuable for probing the biochemical mechanisms of the disease. Available methods for the isolation of bovine liver cells are mostly perfusion‐based and are often time‐consuming, require specialized equipment, or tend to favor the isolation of vascular epithelial cells rather than parenchymal cells. With these considerations in mind, our objective was to develop an in vitro model of FLD, optimizing a non‐perfusion method of hepatic cell isolation from adult cows. Hepatic tissues (~10 g) of cows from the abattoir were immediately collected following slaughter and transported to the laboratory on ice. After several washings with PBS, tissues were chopped, washed, and digested in 200 U/mL of collagenase II for ~30 min with shaking at 37 °C. When hepatic cells were grown, an overnight serum deprivation was done before the treatment with 0.4 mM palmitate, 20 ng/mL TNFα, and a cocktail of 0.4 mM palmitate and 20 ng/mL TNFα. Control cells were grown in growth medium alone. Identification of hepatic cells in culture was done by using immunolocalization of two epithelial cell markers namely, e‐cadherin and mesenchymal marker alpha smooth muscle actin (αSMA). Cell viability, apoptosis, and lipid accumulation were estimated by using lactate dehydrogenase (LDH) assay, caspase 3/7 assay, and oil red O staining, respectively. Hepatic cells were grown after 7–10 days of culture. Mixed cultures of hepatic cells showed presence of e‐cadherin positive hepatocytes and αSMA‐positive stellate cells. Consistent with FLD in vivo, presence of palmitate and palmitate along with TNFα increased (p < 0.05) LDH leakage in comparison to control culture. Likewise, TNFα and palmitate increased (p < 0.05) accumulation of lipid within the cell. In comparison to control cells, presence of palmitate and TNFA enhanced (3–3.5‐fold; p < 0.05) expression of caspase 3/7. Based on preliminary data presented herein, it appears that the non‐perfusion method of hepatic cell isolation and treating primary hepatic cells with palmitate and TNFα is an in vitro model for FLD.