Each cryovial of PHH was thawed and plated according to Lonza’s “Suspension and plateable cryopreserved hepatocytes: technical information and instructions.”. the protocol was followed stepwise minutely, using the recommended thawing and plating media (Lonza, MCHT50 and MP250, respectively). The cells were dispensed and mixed using only wide orifice tips (Rainin, Ref. 17014297). For efficient cell seeding densities and attachment, cells were counted using Trypan Blue Exclusion Method and seeded into Collagen-I coated plates at a density of approximately 100,000 cells/cm2 following the Lonza’s instructions (Lonza, “Suspension and Plateable Cryopreserved Hepatocytes Technical Information and Instructions”). Six hours post seeding, cells were washed with 1 mL of pre-warmed Maintenance Medium (Lonza, MCHT50) before addition of treatment media. The treatment medium was renewed every 24h for a total incubation period of 72h post-seeding. Free fatty acids (FFA) consisting of a 200 µM mixture of a 2:1 ratio of the unsaturated oleic acid to the saturated palmitic acid were added to the maintenance media, to mimic the levels in human steatosis (Gómez-Lechón et al. 2007). In order to facilitate FFA uptake, pre-bounding of free fatty acids to 1% bovine serum albumin in a 1:5 molar ratio (Sigma-Aldrich) was performed by heating the mixture at 40ºC for 2 hours (Kozyra et al. Sci Rep 2018).