DOI: 10.5061/dryad.qv9s4mwhc
title: A self-purifying microfluidic system for identifying drugs acting against adult schistosomes

description: The discovery of novel antihelmintic molecules to combat the development
 and spread of schistosomiasis, a disease caused by several Schistosoma
 flatworm species, mobilizes significant research efforts worldwide. With a
 limited number of biochemical assays for measuring the viability of adult
 worms, the anti-schistosomicidal activity of molecules is usually
 evaluated by microscopic observation of worm mobility and/or integrity
 upon drug exposure. Even if these phenotypical assays enable multiple
 parameters analysis, they are often conducted during several days and need
 to be associated with image-based analysis to minimized subjectivity. We
 describe here a self-purifying microfluidic system enabling the selection
 of healthy adult worms and the identification of molecules acting
 instantly on the parasite. The worms are assayed in a dynamic environment
 that eliminates unhealthy worms that cannot attach firmly to the chip
 walls prior to being exposed to the drug. The detachment of the worms is
 also used as a second step readout for identifying active compounds. We
 have validated this new fluidic screening approach using the two major
 antihelmintic drugs, Praziquantel and Artemisinin. The reported dynamic
 system is simple to produce and to parallelize. Importantly, it enables
 quick and sensitive detection of antischistosomal compounds in no more
 than one hour. 
description: <strong>Microfluidic chip
 design</strong> The geometrical features of the chips were
 designed by using COMSOL MULTIPHYSICS (Comsol AB, Stockholm, Sweden) to
 obtain a single microfluidic chamber, the fluid flow was calculated with
 the microfluidic module using laminar flow conditions assuming a very low
 Reynolds number (see Supplementary Table S3). The 3D FEM model is made of about 2 106 192 degrees of
 freedom using the predefined ‘extra fine’ mesh refinement. In the fluid
 flow model, non-slip initial condition was imposed to all surfaces
 corresponding to a solid/liquid interface. A fixed flow rate (between 0.05
 and 3.4 ml.min<sup>-1</sup>) was used for the inlet and a fixed pressure (P
 = 0 Pa) for the outlet. The equations were solved in 900 s requiring 7.5
 Go using an Intel Core i7-7500U CPU cadenced at 2.7 Ghz with 16 Go RAM
 configuration. <strong>Analysis of viability in various media and
 sera</strong> The viability of the worms was
 evaluated by observing the parasites with
 a macroscope (OPTIKA,
 SZ6745TR, Italy) and using
 a visual ’severity score’ to determine health status. We have defined a 3 levels scale: S2 score is
 for worms having a normal movement, gut peristalsis and normal tissues and
 tegument integrity, and attachment to plate with sucker; S1 score
 corresponds to worms with reduced movement but still gut peristalsis
 and/or degraded tissues or tegument; while S0 corresponds to dead worms
 having no movement or with strong tissue degradation. Each condition was observed for at least 30 s.
 Data were corrected when an S0 score (dead) was followed by an S1 or S2
 score (still alive) and thus to avoid falsely counting worms that were
 still alive as dead. The data are presented as percentages, mean values
 and standard deviations. Statistical significance was calculated with
 ANOVA followed by Tukey’s test with an α risk of 5% (p value &lt;
 0.05) between human, calf and horse sera. Assumptions of ANOVA were
 verified by using a plot of residuals in function of predictions and a
 normal qq-plot. All these data analyses were done using R Software (R Core
 Team (R Foundation for Statistical Computing, Vienna, Austria, 2022) in
 the RStudio environment (1.1.463 release, RStudio Inc., Boston,
 USA). <strong>Adhesion studies on different surfaces</strong>
 For each experiment, 30 couples were introduced
 into the chips with different glass surface coatings: w/o coating, coated
 with MDCK cells, with type I-A collagen or with both MDCK cells and type
 I-A collagen. Chips were placed on a heating block set at 37°C (Major
 Science, MD-02D, USA), and connected to 15 mL tubes placed in a water bath
 set at 37°C. A camera (Microsoft, Q2F-00015, USA) was
 used to monitor the microfluidic channel at the
 beginning of the experiment in order to identify worms that lost adherence
 and to measure the duration of their residence in the microsystem before
 being flushed out by the flow. To modulate flow conditions, the
 peristaltic pump was
 programmed to pull the culture medium at different flow rates: 1) 50
 μL.min<sup>-1</sup> to 300 μL.min<sup>-1</sup> with a step of 50 μL.min<sup>-1</sup> every min; 2) 300 μL.min<sup>-1</sup> to 1 mL.min<sup>-1</sup> with a step of 100 μL.min<sup>-1</sup> every 2 min; 3) 1 mL.min<sup>-1</sup> to 3.4 mL.min<sup>-1</sup> with a step of 200 μL.min<sup>-1</sup> every 3 min. The percentages of worms still attached to the
 chip were plotted against the flow rate. A 2-segment linear regression analysis was
 performed using SigmaPlot v14.5 with Piecewise fitting curve option to
 identify the transition value (T1). We next performed a linear regression
 using the data between 0 and 1.6 mL.min-1 for glass, MDCK and collagen +
 MDCK, while the data between 500 µL.min<sup>-1</sup> and 1.6 mL.min<sup>-1</sup> values were used for collagen. This analysis
 furnished the slope of the first segments, the standard deviation and the
 95% confidence intervals. Experiments were performed in duplicate (n = 2)
 with worms from the same age after perfusion and a 48h maximum duration of
 culture. To compare gripping between males, females or couples, 30 worms
 or couples were introduced in a collagen-coated chip and were perfused at
 increasing flow rates as described above. Experiments were performed in
 triplicate (n=3). Statistical significance was calculated with
 ANOVA followed by Tukey’s test with an α risk of 5% (p-value &lt;
 0.05) between males, females and/or couples. Assumptions of ANOVA were
 verified by using a plot of residuals in function of predictions and a
 normal qq-plot (see source data files). <strong>Drug testing under flow conditions</strong>
 Praziquantel (PZQ) (SIGMA,
 P-4668, USA) was prepared as a 0.5 M stock solution in DMSO and diluted in
 RPMI Horse serum from 900 nM to 25 nM. Artemisinin (ART) (Tokyo chemical
 industry, A2118, Japan) was prepared as a 0.1 M stock solution in DMSO and
 diluted from 900 µM to 12.5 µM in RPMI Horse serum. Stock solutions were
 stored at 4 °C. Once the
 worms were introduced in the system as previously described,
 the flow rate was increased up to 1
 mL.min<sup>-1</sup> by 200 µL.min<sup>-1</sup>
 increments every 5 s in order to favor worm attachment to the
 collagen-coated chips. The recording was started and the 1 mL.min-1 flow
 was maintained for 5 min. This lag period allowed the elimination of
 weakened worms. Next, PZQ or ART was added to the tube containing the
 culture medium. The medium was mixed and infused into the device. The
 video recordings were used to count the worm couples present in the
 microfluidic chamber as a function of time. Experiments were performed in
 triplicate (n = 3) with
 worms from the same age after perfusion and with a maximum duration of
 culture prior experiment of 48h.
 <strong>Drug testing
 under static conditions</strong>
 Typically, 10 worm couples
 were incubated in 12 well plates at 37°C for 120 h in RPMI Horse
 supplemented with PZQ or ART (0 to 900 nM for PZQ or 0 to 900 µM for ART,
 3 mL per well). The media were removed and renewed every 24 h. After this
 step, each well was immediately recorded for 15 s under a
 macroscope (OPTIKA, SZ6745TR, Italy) equipped with a
 camera (Microsoft, Q2F-00015, USA). In this experiment, we used a more
 resolved ‘severity score’ than those classically used for drug evaluation
 against helminths in static conditions (35). We designed for this assay a 5-level scoring
 since it gives a more precise evaluation of phenotypes, in particular
 paralysis or tegument alterations (S4 = Normal mobility and bloodsucker
 adhesion; S3 = reduction of mobility and/or loss of bloodsucker adhesion;
 S2 = minimal mobility or occasional movements; S1 = no mobility except
 intestinal movement; S0 = total loss of mobility, no movement, death). Experiments were performed at least in
 triplicate for each concentration (n &gt; 3) with freshly perfused
 worms (&lt; 12h).
description: <strong>Programs required to visualize and analyse
 data:</strong> Videos: mp4 file player
 Tables: Excel 2019 or any software compatible .xlsx
 file Scripts: Rstudio (1.1463)
 Fluidic simulations: Comsol Multiphysics (v.5.6)
 IC50 data and graphs: SigmaPlot (v.15)