Functionality of primary hepatic non-parenchymal cells in a 3D spheroid model and contribution to acetaminophen hepatotoxicity

Abstract

In addition to hepatocytes, the liver comprises a host of specialised non-parenchymal cells which are important to consider in the development of in vitro models which are both physiologically and toxicologically relevant. We have characterized a 3D co-culture system comprising primary human hepatocytes (PHH) and non-parenchymal cells (NPC) and applied it to the investigation of acetaminophen-induced toxicity. Firstly, we titrated ratios of PHH:NPC and confirmed the presence of functional NPCs via both immunohistochemistry and activation with both LPS and TGF-β. Based on these data we selected a ratio of 2:1 PHH:NPC for further studies. We observed that spheroids supplemented with NPCs were protected against acetaminophen (APAP) toxicity as determined by ATP (up to threefold difference in EC 50 at day 14 compared to hepatocytes alone) and glutathione depletion, as well as miR-122 release. APAP metabolism was also altered in the presence of NPCs, with significantly lower levels of APAP-GSH detected. Expression of several CYP450 enzymes involved in the bioactivation of APAP was also lower in NPC-containing spheroids. Spheroids containing NPCs also expressed higher levels of miRNAs which have been implicated in APAP-induced hepatotoxicity, including miR-382 and miR-155 which have potential roles in liver regeneration and inflammation, respectively. These data indicate that the interaction between hepatocytes and NPCs can have significant metabolic and toxicological consequences important for the correct elucidation of hepatic safety mechanisms.

Current Perspective: 3D Spheroid Models Utilizing Human-Based Cells for Investigating Metabolism-Dependent Drug-Induced Liver Injury

“…Other cell types used in combination with hepatocytes to establish in-vitro liver models include the non-parenchymal cell types found in the liver (i.e., Kupffer cells, stellate cells, liver sinusoidal endothelial cells (LSECs), and cholangiocytes), with the aim of recapitulating the liver's multi-cellular environment and paracrine signaling. Similar to PHHs, NPCs can be primary human cells derived from liver resection/biopsies (e.g., NPC liver fractions or isolated Kupffer cells) (32,68,(78)(79)(80)(81). Cancer/ immortalized cell lines can also be used to represent the liver-specific NPCs (82).…”

“…Endothelial cells and mesenchymal stem cells are also being used in combination with hepatocytes to try to emulate the endodermal, endothelial, and mesenchymal interactions that occur during organogenesis and liver bud formation (83), with human umbilical vein endothelial cells (HUVECs) being used (83,84) or endothelial cells derived from pluripotent stem cells (72,85). Importantly, data indicates that the addition of these different cell types can promote hepatocyte maturation and metabolic competence in vitro (32,68,71,72,(78)(79)(80)(82)(83)(84)(85)(86) and, therefore, may be important for detecting some mechanisms of DILI.…”

“…Indeed, 3D coculture models have been established entirely from immortalized / cancer-derived cell lines (i.e., HepaRG cells, hTERT-hepatic stellate cells, and THP-1 monocytes) (82,118), entirely from primary human cells (e.g., PHHs, Kupffer cells, stellate cells and cholangiocytes) (32,68,(78)(79)(80)(81), and entirely from the same iPSC (i.e., an isogenic model) (85). The addition of Kupffer cells and stellate cells provides a convenient model to study DILI with an immune-mediated mechanism of toxicity (78), investigate the roles of these cell types in drug-induced toxicities (82) and evaluate the pro-inflammatory and pro-fibrotic potential of drugs (68,80). Cocultures that include just PHHs and Kupffer cells have also been described and used to evaluate the influence of Kupffer cell activation on a drug's toxic potential, with data suggesting that they can exacerbate toxicity for some drugs and be protective for others (81).…”

Utility of Three-Dimensional Cultures of Primary Human Hepatocytes (Spheroids) as Pharmacokinetic Models

“…In this context, it has been reported that the hepatic phenotype of 3D-cultured spheroids is well maintained during long-term culture (for at least 35 days), as evidenced by well-sustained ATP levels, albumin secretion, and the overall stability of CYP enzyme activities [ 46 ]. Moreover, Bell et al evaluated acetaminophen-induced hepatotoxicity using a 3D co-culture system of PHHs and non-parenchymal cells, noting that high-level expression of miRNAs involved in the toxicity, i.e., mi-382 and mi-155, was observed [ 47 ]. In addition, to evaluate the potential of their system as a predictive model for drug-induced hepatotoxicity, they compared three emerging cell systems: hepatocytes derived from induced pluripotent stem cells, HepaRG cells, and PHH spheroids, at the transcriptional and functional levels in a multicenter study [ 48 ].…”

“…Cultured human cancer-derived hepatocytes have long been used for human-specific toxicity assessments, but they generally have lower expression levels of drug-metabolizing enzymes than PHHs [ 39 , 40 ]. Various applications of PHH spheroids as pharmacokinetic models [ 41 , 42 , 43 , 44 ] or hepatotoxicity detection models [ 16 , 45 , 46 , 47 , 48 , 49 , 50 , 51 ] have already been reported, and it has become clear that this model is extremely versatile and offers a variety of advantages over 2D cultures ( Table 2 ) [ 16 , 41 , 42 , 43 , 44 , 45 , 46 , 47 , 48 , 49 , 50 , 51 , 52 , 53 , 54 , 55 , 56 , 57 ]. PHH spheroids have also been used as disease models [ 58 , 59 , 60 ].…”