Modeling of intravenous caspofungin administration using an intestine-on-chip reveals altered Candida albicans microcolonies and pathogenicity.

Candida albicans is a commensal yeast of the human intestinal microbiota that, under predisposing conditions, can become pathogenic and cause life-threatening systemic infections (candidiasis). Fungal-host interactions during candidiasis are commonly studied using conventional 2D in vitro models, which have provided critical insights into the pathogenicity. However, microphysiological models with a higher biological complexity may be more suitable to mimic in vivo-like infection processes and antifungal drug efficacy. Therefore, a 3D intestine-on-chip model was used to investigate fungal-host interactions during the onset of invasive candidiasis and evaluate antifungal treatment under clinically relevant conditions. By combining microbiological and image-based analyses we quantified infection processes such as invasiveness and fungal translocation across the epithelial barrier. Additionally, we obtained novel insights into fungal microcolony morphology and association with the tissue. Our results demonstrate that C. albicans microcolonies induce injury to the epithelial tissue by disrupting apical cell-cell contacts and causing inflammation. Caspofungin treatment effectively reduced the fungal biomass and induced substantial alterations in microcolony morphology during infection with a wild-type strain. However, caspofungin showed limited effects after infection with an echinocandin-resistant clinical isolate. Collectively, this organ-on-chip model can be leveraged for in-depth characterization of pathogen-host interactions and alterations due to antimicrobial treatment.

Evaluation of drug-induced liver toxicity of trovafloxacin and levofloxacin in a human microphysiological liver model.

Drug-induced liver injury induced by already approved substances is a major threat to human patients, potentially resulting in drug withdrawal and substantial loss of financial resources in the pharmaceutical industry. Trovafloxacin, a broad-spectrum fluoroquinolone, was found to have unexpected side effects of severe hepatotoxicity, which was not detected by preclinical testing. To address the limitations of current drug testing strategies mainly involving 2D cell cultures and animal testing, a three-dimensional microphysiological model of the human liver containing expandable human liver sinusoidal endothelial cells, monocyte-derived macrophages and differentiated HepaRG cells was utilized to investigate the toxicity of trovafloxacin and compared it to the structurally-related non-toxic drug levofloxacin. In the model, trovafloxacin elicited vascular and hepatocellular toxicity associated with pro-inflammatory cytokine release already at clinically relevant concentrations, whereas levofloxacin did not provoke tissue injury. Similar to in vivo, cytokine secretion was dependent on a multicellular immune response, highlighting the potential of the complex microphysiological liver model for reliably detecting drug-related cytotoxicity in preclinical testing. Moreover, hepatic glutathione depletion and mitochondrial ROS formation were elucidated as intrinsic toxicity mechanisms contributing to trovafloxacin toxicity.

Generation & characterization of expandable human liver sinusoidal endothelial cells and their application to assess hepatotoxicity in an advanced in vitro liver model.

Liver sinusoidal endothelial cells (LSECs) are highly specialized endothelial cells forming the hepatic sinusoidal wall. Besides their high endocytic potential, LSECs have been demonstrated to markedly contribute to liver homeostasis and immunity, and may partially explain unexpected hepatotoxicity of drug candidates. However, their use for in vitro investigations is compromised by poor cell yields and a limited proliferation capacity. Here, we report the transient expansion of primary human LSECs from three donors by lentiviral transduction. Transduced ("upcyte®") LSECs were able to undergo at least 25 additional population doublings (PDs) before growth arrest due to senescence. Expanded upcyte® LSECs maintained several characteristics of primary LSECs, including expression of surface markers such as MMR and LYVE-1 as well as rapid uptake of acetylated LDL and ovalbumin. We further investigated the suitability of expanded upcyte® LSECs and proliferating upcyte® hepatocytes for detecting acetaminophen toxicity at millimolar concentrations (0, 0.5, 1, 2, 5, 10 mM) in static 2D cultures and a microphysiological 3D model. upcyte® LSECs exhibited a higher sensitivity to acetaminophen-induced toxicity compared to upcyte® hepatocytes in 2D culture, however, culturing upcyte® LSECs together with upcyte® hepatocytes in a co-culture reduced APAP-induced toxicity compared to 2D monocultures. A perfused Dynamic42 3D model was more sensitive to acetaminophen than the 2D co-culture model. Cytotoxicity in the 3D model was evident by decreased cellular viability, elevated LDH release, reduced nuclei counts and impaired cell morphology. Taken together, our data demonstrate that transient expansion of LSECs represents a suitable method for generation of large quantities of cells while maintaining many characteristics of primary cells and responsiveness to acetaminophen.