Here, we introduce a human liver microtissue model in a 96-well format composed of cryopreserved primary human hepatocytes in combination with non-parenchymal cells (Kupffer and endothelial cells) and its use for long-term testing and inflammation-mediated toxicity (3D Insight™ Human Liver Microtissues). The accumulation of hepatocytes and non-parenchymal cells in hanging drops resulted in microtissue formation within 3 days (Fig. 1a). After microtissue formation, the spheroids were either harvested for histological analysis or transferred into a non-adhesive spheroid-specific 96-well plate for long-term culture and drug treatment (Fig. 1b–d). Immunohistochemical staining for the epithelial marker cytokeratins 8 (CK8) reveals an intact cellular phenotype, indicates direct cell–cell contacts and the typical polygonal, bicuboidal shape of hepatocytes (Fig. 2a). Kupffer cell populations were distributed throughout the microtissue and were observed by CD68 staining similar to endothelial cells positive for CD31 (Fig. 2b, c). The macrophages exhibited typical morphology with elongated shapes. Glycogen storage capability was confirmed by periodic acid schiff staining (Fig. 2d, dark violet stain). The presence of transporters was exemplified by staining for the multidrug resistance protein 1 (MDR1) and bile salt export pump (BSEP) (Fig. 2e, f). These transporters are ATP-dependent drug efflux pumps mediating transport of endogenous and xenobiotic substances. The transporters are clearly expressed in a polarized manner on the apical surface of the primary hepatocytes (Fig. 2e, f). Their staining pattern indicates presence of bile canaliculi, into which hepatocytes secrete their metabolized toxic products. Some of the bile canaliculi appear to be open to the outer surface of the hepatosphere, as highlighted by MDR1 staining (Fig. 2e). Liver microtissues remained stable over 5 weeks in culture as shown by a constant ATP content (Fig. 3a). This extended life span compared to 2D cultures of hepatocytes is most likely due to extensive cell–cell contacts, which are essential for maintaining the differentiated status of hepatocytes. Besides the stable viability, functionality of liver microtissues is preserved over 5 weeks, as indicated by persistent albumin secretion (Fig. 3b).
The prolonged hepatocyte lifetime and functionality in comparison with conventional 2D culture of hepatocytes allows for long-term studies with repeated dosing to evaluate chronic hepatotoxic effects. Two hepatotoxic compounds acetaminophen and diclofenac were tested with respect to their long-term toxicological profile. Acetaminophen is the major cause of DILI in humans, although toxicity is dose-dependent and varies between patient populations (Stine and Lewis 2011). At therapeutic doses, a proportion of the drug undergoes bio-activation by CYP2E1, CYP1A2 and CYP3A4. The reactive intermediate depletes intracellular glutathione pools leading to hepatocyte cell death (Park et al. 2005). So far, 2D cultures of hepatocytes have not been able to convincingly recapitulate acetaminophen-induced toxicity in vitro (Fey and Wrzesinski 2012). Treatment of liver microtissues over 14 days with 3 re-dosing’s resulted in a concentration-dependent increasing cell death with an IC50 value of 754.2 μM (Fig. 4a). Diclofenac is a non-steroidal anti-inflammatory drug that has a strong association with hepatotoxicity. The mechanism is thought to involve phase I enzyme activity (multiple P450-catalyzed oxidations), phase II enzyme activity (glucoronylation) and mechanism-based inhibition (Park et al. 2005). In comparison with 2D cultures of human hepatocytes (calculated IC50 value of 331 μM) (Bort et al. 1999), long-term treated liver microtissues displayed an increased sensitivity toward this drug with an IC50 value of 178.6 μM (Fig. 4b).
Most directly hepatotoxic compounds are detected during pre-clinical investigations. However, indirectly hepatotoxic compounds involving the immune system are not detected during pre-clinical phases, such as trovafloxacin (Shaw et al. 2010). Recent animal experiments indicated that trovafloxacin is only hepatotoxic in combination with an inflammatory stimulus, such as lipopolysaccharide (LPS) or TNF-α (Shaw et al. 2007, 2010; Liguori et al. 2010). The mechanism is thought to involve enhanced cytokine secretion and accumulation in the liver, causing caspase activation and subsequent liver injury. Induction of the inflammatory response in liver microtissues by LPS resulted in elevated levels of IL-6 secretion, verifying the responsiveness of incorporated macrophages in the liver microtissues (Fig. 4c). The addition of LPS shifted the hepatotoxic threshold of trovafloxacin about threefold from 220 (without LPS) to 71 μM in the presence of LPS (Fig. 4d).
Developed to overcome the limitations of conventional 2D culture, multi-cell type 3D liver microtissues resemble liver-like cell composition and an extended stability in culture. The long-term viability and functionality of liver microtissues allows for routine compound testing as well as chronic and inflammation-mediated toxicity. The 96-well format allows for microtissue mass production enabling the implementation of an organotypic liver model at an early time point in drug development.