High throughput transepithelial electrical resistance (TEER) measurements on perfused membrane-free epithelia.

Assessment of epithelial barrier function is critically important for studying healthy and diseased biological models. Here we introduce an instrument that measures transepithelial electrical resistance (TEER) of perfused epithelial tubes in the microfluidic OrganoPlate platform. The tubules are grown in microfluidic channels directly against an extracellular matrix, obviating the need for artificial filter membranes. We present TEER measurements on Caco-2 intestinal and renal proximal tubule epithelium. Forty tubules on one single plate were interrogated in less than a minute. We show that TEER measurement is significantly more sensitive than a fluorescent reporter leakage assay in response to staurosporine. We demonstrate a 40-channel time-lapse data acquisition over a 25 hour time period under flow conditions. We furthermore observed a 50% reduction in Caco-2 TEER values following exposure to a cocktail of inflammatory cytokines. To our best knowledge, this is the first instrument of its kind that allows routine TEER studies in perfused organ-on-a-chip systems without interference by artificial filter membranes. We believe the apparatus will contribute to accelerating routine adoption of perfused organ-on-a-chip systems in academic research and in industrial drug development.

Combining Extracellular miRNA Determination with Microfluidic 3D Cell Cultures for the Assessment of Nephrotoxicity: a Proof of Concept Study.

Drug-induced kidney injury is often observed in the clinics and can lead to long-term organ failure. In this work, we evaluated a novel in vitro system that aims at detecting whether compounds can cause renal proximal tubule damage in man. For this, we implemented organotypic cultures of human conditionally immortalized proximal tubule epithelial cells overexpressing the organic anion transporter 1 (ciPTEC-OAT1) in a three-channel OrganoPlate under microfluidic conditions. Cells were exposed to four known nephrotoxicants (cisplatin, tenofovir, cyclosporine A, and tobramycin). The effect on cell viability and NAG release into the medium was determined. A novel panel of four miRNAs (mir-21, mir-29a, mir-34a, and mir-192) was selected as potential biomarkers of proximal tubule damage. After nephrotoxicant treatment, miRNA levels in culture medium were earlier indicators than cell viability (WST-8 assay) and outperformed NAG for proximal tubule damage. In particular, mir-29a, mir-34a, and mir-192 were highly reproducible between experiments and across compounds, whereas mir-21 showed more variability. Moreover, similar data were obtained in two different laboratories, underlining the reproducibility and technical transferability of the results, a key requirement for the implementation of novel biomarkers. In conclusion, the selected miRNAs behaved like sensitive biomarkers of damage to tubular epithelial cells caused by several nephrotoxicity mechanisms. This biomarker panel, in combination with the 3D cultures of ciPTEC-OAT1 in the OrganoPlate, represents a novel tool for in vitro nephrotoxicity detection. These results pave the way for the application of miRNAs in longitudinal, time-course in vitro toxicity studies.

96 perfusable blood vessels to study vascular permeability in vitro.

Current in vitro models to test the barrier function of vasculature are based on flat, two-dimensional monolayers. These monolayers do not have the tubular morphology of vasculature found in vivo and lack important environmental cues from the cellular microenvironment, such as interaction with an extracellular matrix (ECM) and exposure to flow. To increase the physiological relevance of in vitro models of the vasculature, it is crucial to implement these cues and better mimic the native three-dimensional vascular architecture. We established a robust, high-throughput method to culture endothelial cells as 96 three-dimensional and perfusable microvessels and developed a quantitative, real-time permeability assay to assess their barrier function. Culture conditions were optimized for microvessel formation in 7 days and were viable for over 60 days. The microvessels exhibited a permeability to 20 kDa dextran but not to 150 kDa dextran, which mimics the functionality of vasculature in vivo. Also, a dose-dependent effect of VEGF, TNFα and several cytokines confirmed a physiologically relevant response. The throughput and robustness of this method and assay will allow end-users in vascular biology to make the transition from two-dimensional to three-dimensional culture methods to study vasculature.

Mitotic catastrophe triggered in human cancer cells by the viral protein apoptin.

Mitotic catastrophe is an oncosuppressive mechanism that senses mitotic failure leading to cell death or senescence. As such, it protects against aneuploidy and genetic instability, and its induction in cancer cells by exogenous agents is currently seen as a promising therapeutic end point. Apoptin, a small protein from Chicken Anemia Virus (CAV), is known for its ability to selectively induce cell death in human tumor cells. Here, we show that apoptin triggers p53-independent abnormal spindle formation in osteosarcoma cells. Approximately 50% of apoptin-positive cells displayed non-bipolar spindles, a 10-fold increase as compared to control cells. Besides, tumor cells expressing apoptin are greatly limited in their progress through anaphase and telophase, and a significant drop in mitotic cells past the meta-to-anaphase transition is observed. Time-lapse microscopy showed that mitotic osteosarcoma cells expressing apoptin displayed aberrant mitotic figures and/or had a prolonged cycling time during mitosis. Importantly, all dividing cells expressing apoptin eventually underwent cell death either during mitosis or during the following interphase. We infer that apoptin can efficiently trigger cell death in dividing human tumor cells through induction of mitotic catastrophe. However, the killing activity of apoptin is not only confined to dividing cells, as the CAV-derived protein is also able to trigger caspase-3 activation and apoptosis in non-mitotic cancer cells.