2.3. Culture Media

Williams medium E with Glutamax at pH 7.4 (Invitrogen, Bleiswijk, The Netherlands), supplemented with gentamycin (50mg/mL; Invitrogen) and L-carnitine (1 mM, Sigma-Aldrich, St. Louis, MO, USA), was used as control medium. To mimic metabolic syndrome, a culture medium with additional supraphysiological concentrations (Table 1, unless indicated otherwise) of glucose (Merck, Darmstadt, Germany), fructose (Merck), human insulin (Sigma-Aldrich), palmitic acid and oleic acid (Sigma-Aldrich) was used. Concentrations were based on human serum concentrations [36,37] and in vivo rodent portal vein concentrations [34,38] and optimised for this study.

Composition of culture media.

CTR = control; GFI = glucose, fructose and insulin; GFIPO = glucose, fructose, insulin, palmitic acid and oleic acid.

Palmitic acid and oleic acid were dissolved in 0.1 M sodium hydroxide (Merck, Darmstadt, Germany) at 70 °C, and then mixed with preheated 0.04% BSA solution (Sigma-Aldrich, St. Louis, MO, USA) at 55 °C. The same concentration of BSA and sodium hydroxide was added to control media. Addition of this amount of BSA and sodium hydroxide did not affect medium pH and PCLS viability.

Sodium Butyrate (NaB; Sigma-Aldrich) and Sodium chloride (NaCl; Sigma-Aldrich) were dissolved in PBS (Gibco, ThermoFisher Scientific, Waltham, MA, USA). NaCl was used as a control for the NaB addition and is presented as NaCl throughout the paper to avoid confusion with the control medium CTR (Table 1). The concentration of NaB or NaCl in medium was 1mM, unless specified otherwise.