Real-time and automated monitoring of antischistosomal drug activity profiles for screening of compound libraries.

Schistosomiasis is a neglected tropical disease that affects over 200 million people annually. As the antischistosomal drug pipeline is currently empty, repurposing of compound libraries has become a source for accelerating drug development, which demands the implementation of high-throughput and efficient screening strategies. Here, we present a parallelized impedance-based platform for continuous and automated viability evaluation of Schistosoma mansoni schistosomula in 128 microwells during 72 h to identify antischistosomal hits in vitro. By initially screening 57 repurposed compounds against larvae, five drugs are identified, which reduce parasite viability by more than 70%. The activity profiles of the selected drugs are then investigated via real-time dose-response monitoring, and four compounds reveal high potency and rapid action, which renders them suitable candidates for follow-up tests against adult parasites. The study shows that our device is a reliable tool for real-time drug screening analysis of libraries to identify new promising therapeutics against schistosomiasis.

What is the future of electrical impedance spectroscopy in flow cytometry?

More than 20 years ago, electrical impedance spectroscopy (EIS) was proposed as a potential characterization method for flow cytometry. As the setup is comparably simple and the method is label-free, EIS has attracted considerable interest from the research community as a potential alternative to standard optical methods, such as fluorescence-activated cell sorting (FACS). However, until today, FACS remains by and large the laboratory standard with highly developed capabilities and broad use in research and clinical settings. Nevertheless, can EIS still provide a complement or alternative to FACS in specific applications? In this Perspective, we will give an overview of the current state of the art of EIS in terms of technologies and capabilities. We will then describe recent advances in EIS-based flow cytometry, compare the performance to that of FACS methods, and discuss potential prospects of EIS in flow cytometry.

Evaluation of Human Liver Microtissues for Drug Screening on Schistosoma mansoni Schistosomula.

Schistosomiasis is a major neglected tropical disease with more than 200 million infections annually. Despite only one drug, praziquantel, being available, the drug pipeline against schistosomiasis is empty, and drug screening tools have limitations. We evaluated the potential of human liver microtissues (hLiMTs) in antischistosomal drug discovery. Because hLiMTs express all human P450 enzymes, they are an excellent tool to evaluate compounds' bioinactivation, bioactivation, and toxicity. To validate the metabolic conversion capacity of hLiMTs, we first quantified (R)- and (S)-praziquantel and the main metabolite trans-OH-praziquantel following incubation with 0.032-50 µM (0.01-15.62 µg/mL) praziquantel for up to 72 h by a validated LC-MS/MS method. We cocultured hLiMTs with newly transformed schistosomula (NTS) and evaluated the antischistosomal activity and cytotoxicity of three prodrugs terfenadine, tamoxifen citrate, and flutamide. HLiMTs converted 300-350 ng (R)-praziquantel within 24 h into trans-OH-praziquantel. We observed changes in the IC50 values for terfenadine, flutamide, and tamoxifen citrate in comparison to the standard NTS assay in vitro. Cytotoxicity was observed at high concentrations of flutamide and tamoxifen citrate. An in vitro platform containing hLiMTs could serve as an advanced drug screening tool for Schistosoma mansoni, providing information on reduced or increased activity and toxicity.

Parallelized Wireless Sensing System for Continuous Monitoring of Microtissue Spheroids.

Currently, the use of electrical readout methods for the investigation of microtissue spheroids in combination with lab automation tools is hindered by the cable connections that are required to interrogate the on-chip-integrated electrodes. To overcome this limitation, we developed a wireless sensor scheme, which can detect the size variation of microtissues during long-term culturing and drug exposure assays. The sensor system includes an interrogation board, which is composed of an inductor-capacitor (LC) readout circuit, and the tissue culture platform with integrated split-ring sensors. The magnetic coupling between the LC circuit and the sensors enables the interrogation of the on-chip sensors without any wire connection to the culture platform. By optimizing the sensor dimensions and the LC resonance frequencies, we were able to avoid cross talk between neighboring sensors. We integrated 12 tissue compartments on a standard microscopy slide with a sensor-to-sensor pitch of 9 mm, which is in accordance with standard 96-well plate dimensions. As a proof-of-concept experiment for the developed system, we monitored continuously and during more than four days the growth inhibition of colon cancer microtissue spheroids that had been exposed to different concentrations of doxorubicin, a chemotherapeutic compound. The stability of the measurements during long-term culturing and the compatibility of the sensor scheme with standard lab equipment offer great potential for automated electrical microtissue spheroid characterization.

Parallelized Impedance-Based Platform for Continuous Dose-Response Characterization of Antischistosomal Drugs.

Schistosomiasis is an acute and chronic disease caused by tropical parasitic worms of the genus Schistosoma, which parasitizes annually over 200 million people worldwide. Screening of antischistosomal compounds is hampered by the low throughput and potential subjectivity of the visual evaluation of the parasite phenotypes, which affects the current drug assays. Here, an impedance-based platform, capable of assessing the viability of Schistosoma mansoni schistosomula exposed to drugs, is presented. This automated and parallelized platform enables unbiased and continuous measurements of dose-response relationships for more than 48 h. The platform performance is established by exposure of schistosomula to three test compounds, praziquantel, oxethazaine, and mefloquine, which are known to affect the larvae phenotypes. The system is thereafter used to investigate the response of schistosomula to methiothepine, an antipsychotic compound, which causes complex drug-induced effects. Continuous monitoring of the parasites reveals transient behavioral phenotypes and allows for extracting temporal characteristics of dose-response curves, which are essential for selecting drugs that feature high activity and fast kinetics of action. These measurements demonstrate that impedance-based detection provides a wealth of information for the in vitro characterization of candidate antischistosomals and, represents a promising tool for the identification of new lead compounds.

Effect of nanosecond pulsed electric field treatment on cell proliferation of microalgae.

Photoautotrophic microalgae based biorefinery concepts are currently not competitive compared to other established production systems. Therefore, innovative upstream processes need to be developed to increase the competitiveness of photoautotrophic microalgae biorefinery concepts. Abiotic sub-lethal stress induction via nanosecond pulsed electric field (nsPEF) treatment might be a viable process to increase the efficiency of photoautotrophic microalgae cultivation. In this work, an increased cell growth after nsPEF treatment was observable. Application of nsPEF to highly proliferating cells in a repetitive process resulted in a statistical significant increase in cell growth (p = 0.009). The effect was most pronounced after five days wherefore cellular structures and processes were analyzed to reveal a possible mechanism. Within this work, a protocol for increased cell proliferation with a possible mechanism was derived, which improves competitiveness of photoautotrophic microalgae biorefineries in the future. However, based on the derived mechanism, the results are also relevant for other microorganisms.

Impedance-Based Microfluidic Assay for Automated Antischistosomal Drug Screening.

Schistosomiasis is a neglected tropical disease, caused by parasitic worms, which affects almost 200 million people worldwide. For over 40 years, chemotherapeutic treatment has relied on the administration of praziquantel, an efficacious drug against schistosomiasis. However, concerns about developing drug resistance require the discovery of novel drug compounds. Currently, the drug-screening process is mostly based on the visual evaluation of drug effects on worm larvae in vitro by a trained operator. This manual process is extremely labor-intensive, has limited throughput, and may be affected by subjectivity of the operator evaluation. In this paper, we introduce a microfluidic platform with integrated electrodes for the automated detection of worm larvae viability using an impedance-based approach. The microfluidic analysis unit consists of two sets of electrodes and a channel of variable geometry to enable counting and size detection of single parasite larvae and the collective evaluation of the motility of the larvae as an unbiased estimator for their viability. The current platform also allows for multiplexing of the analysis units resulting in increased throughput. We used our platform to record size and motility variations of Schistosoma mansoni larvae exposed to different concentrations of mefloquine, a drug with established in vitro antischistosomal properties. The developed platform demonstrates the potential of integrated microfluidic platforms for high-throughput antischistosomal drug screening.