Advances in organ-on-chip technologies for the application in in vitro drug development provide an attractive alternative approach to replace ethically controversial animal testing and to establish a basis for accelerated drug development. In recent years, various chip-based tissue culture systems have been developed, which are mostly optimized for cultivation of one single cell type or organoid structure and lack the representation of multi organ interactions. Here we present an optimized microfluidic chip design consisting of interconnected compartments, which provides the possibility to mimic the exchange between different organ specific cell types and enables to study interdependent cellular responses between organs and demonstrate that such tandem system can greatly improve the reproducibility and efficiency of toxicity studies. In a simplified liver-kidney-on-chip model, we showed that hepatic cells that grow in microfluidic conditions abundantly and stably expressed metabolism-related biomarkers. Moreover, we applied this system for investigating the biotransformation and toxicity of Aflatoxin B1 (AFB1) and Benzoalphapyrene (BαP), as well as the interaction with other chemicals. The results clearly demonstrate that the toxicity and metabolic response to drugs can be evaluated in a flow-dependent manner within our system, supporting the importance of advanced interconnected multiorgans in microfluidic devices for application in in vitro toxicity testing and as optimized tissue culture systems for in vitro drug screening.

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Background Aerobic glycolysis, discovered by Otto Warburg, is a hallmark of cancer metabolism even though not yet fully understood. The low activity of the cancerous pyruvate kinase isozyme (M2) is thought to play an important role by facilitating the conversion of glycolytic intermediates to other anabolic pathways to support tumors’ high proliferation rate. Methods Five breast cancer cell lines representing different molecular subtypes were used in this study where real time measurements of cellular bioenergetics and immunoblotting analysis of energy- and nutrient-sensing pathways were employed to investigate the potential effects of PKM2 allosteric activator (DASA-58) in glucose rewiring. Results In this study, we show that DASA-58 can induce pyruvate kinase activity in breast cancer cells without affecting the overall cell survival. The drug is also able to reduce TXNIP levels (an intracellular glucose sensor) probably through depletion of upstream glycolytic metabolites and independent of AMPK and ER signaling. AMPK shows an induction in phosphorylation (T172) in response to treatment an effect that can be potentiated by combining DASA-58 with other metabolic inhibitors. Conclusions Altogether, the multifaceted metabolic reprogramming induced by DASA-58 in breast cancer cells increases their susceptibility to other therapeutics suggesting the suitability of the intracellular glucose sensor TXNIP as a marker of PK activity.

Despite rapid development and deployment of vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), clinically relevant modalities to curb the pandemic by directly attacking the virus on a genetic level remain highly desirable and are urgently needed. Here we comprehensively illustrate the capacity of adeno-associated virus (AAV) vectors co-expressing a cocktail of three short hairpin RNAs (shRNAs; RNAi triggers) directed against the SARS-CoV-2 RdRp and N genes as versatile and effective antiviral agents. In cultured monkey cells and human gut organoids, our most potent vector, SAVIOR (SARS virus repressor), suppressed SARS-CoV-2 infection to background levels. Strikingly, in control experiments using single shRNAs, multiple SARS-CoV-2 escape mutants quickly emerged from infected cells within 24-48 h. Importantly, such adverse viral adaptation was fully prevented with the triple-shRNA AAV vector even during long-term cultivation. In addition, AAV-SAVIOR efficiently purged SARS-CoV-2 in a new model of chronically infected human intestinal cells. Finally, intranasal AAV-SAVIOR delivery using an AAV9 capsid moderately diminished viral loads and/or alleviated disease symptoms in hACE2-transgenic or wild-type mice infected with human or mouse SARS-CoV-2 strains, respectively. Our combinatorial and customizable AAV/RNAi vector complements ongoing global efforts to control the coronavirus disease 2019 (COVID-19) pandemic and holds great potential for clinical translation as an original and flexible preventive or therapeutic antiviral measure.