Introduction: Kupffer cells (KCs) directly interact with hepatocytes during liver inflammation by modifying immunity and xenobiotic metabolism. KCs are known to suppress the expression of multiple cytochrome P450 enzymes following activation with lipopolysaccharide (LPS). Since KCs are involved in liver diseases that are characterized with inflammation including viral and autoimmune hepatitis and non-alcoholic steatohepatitis (NASH), there is a need for a KCs and hepatocyte co-culture model for drug discovery and ADME/Tox applications. This study hypothesizes that the use of cryopreserved rat and human KCs in co-culture with hepatocytes will recreate liver inflammation and model acetaminophen (APAP) cytotoxicity observed during liver inflammation. Methods: Fresh and cryopreserved Kupffer cells were characterized by morphology, plateability, phagocytosis, immunohistochemistry staining for CD68 and CD163, and cytokine release for IL-6 and TNFα by ELISA in response to 24hrs treatment with 1µg/mL LPS. KCs and hepatic co-cultures were cultured in DMEM medium supplemented with non-essential amino acids (NEAA), Insulin-Transferring-Selenium (ITS+) and Penicillin/Streptomycin. For plating and maintenance media, additional 10% or 2% fetal bovine serum (FBS) was used, respectively. Since corticosteroids inhibit inflammation, dexamethasone or hydrocortisone were not used with KCs. For experiments with APAP cytotoxicity, inflammatory ratios of approximately 1:2-1:4 KCs to hepatocytes were used. Co-cultures were cultured for 24 hours and then pre-treated with 1µg/mL LPS for 24, 4 and 0 hours prior to addition of 10mM APAP for additional 24hr in the same media containing LPS. Cells were evaluated for viability using PrestoBlueTM reagent, morphology and cytokine release (e.g. IL-6, TNFα) by ELISA. Real time qRT-PCR was performed to establish gene expression changes in metabolism, inflammation and viability. Results: Fresh and cryopreserved rat KCs were found to be similar after comparison of morphology, plateability, culture purity via immunofluorescent staining. The purity of KCs was found to be at 85.6% (±1.9 SE) for CD68 and at 93.5% (±0.4 SE) for CD163, commonly recognized cell surface markers of KCs. Using cytokine panel, activated fresh and cryopreserved co-cultures revealed comparable upregulated expression of IL-1α, IL-1β and IL-2 by at least two fold, and IL-6 IL-10 and TNFα by at least four fold; conversely, they revealed down regulated expression in IL-4 and IL-12 by at least two fold. For the cytotoxicity experiments, viability and morphology data analysis revealed time-dependent hepato-protection in co-cultures at 4 hours pre-treatment with LPS, but not at 0 and 24 hours. A strong correlation was observed with cytokine release of IL-6 and TNFα, and expression of CYP1A2 and CYP2E1. Conclusions: Cryopreserved KCs can maintain known characteristics and functions as compared to freshly isolated cells. APAP cytotoxicity data show that these cells can be effectively used for modeling liver inflammation and reveal hepato-protective role of KCs as shown at 4 hour pre-treatment with LPS. Cryopreserved KCs when co-cultured with hepatocytes, provide a powerful in vitro ADME/Tox tool to evaluate the effects of xenobiotic metabolism during inflammation.