Microfluidics-based single cell analysis reveals drug-dependent motility changes in trypanosomes.

We present a single cell viability assay, based on chemical gradient microfluidics in combination with optical micromanipulation. Here, we used this combination to in situ monitor the effects of drugs and chemicals on the motility of the flagellated unicellular parasite Trypanosoma brucei; specifically, the local cell velocity and the mean squared displacement (MSD) of the cell trajectories. With our method, we are able to record in situ cell fixation by glutaraldehyde, and to quantify the critical concentration of 2-deoxy-d-glucose required to completely paralyze trypanosomes. In addition, we detected and quantified the impact on cell propulsion and energy generation at much lower 2-deoxy-d-glucose concentrations. Our microfluidics-based approach advances fast cell-based drug testing in a way that allows us to distinguish cytocidal from cytostatic drug effects, screen effective dosages, and investigate the impact on cell motility of drugs and chemicals. Using suramin, we could reveal the impact of the widely used drug on trypanosomes: suramin lowers trypanosome motility and induces cell-lysis after endocytosis.

[1]  M. Nobili,et al.  Brownian Motion of an Ellipsoid , 2006, Science.

[2]  C. Burri,et al.  Treatment of human African trypanosomiasis--present situation and needs for research and development. , 2002, The Lancet. Infectious diseases.

[3]  Gwo-Bin Lee,et al.  A microfluidic cell culture platform for real-time cellular imaging , 2009, Biomedical microdevices.

[4]  H Hirumi,et al.  Continuous cultivation of Trypanosoma brucei blood stream forms in a medium containing a low concentration of serum protein without feeder cell layers. , 1989, The Journal of parasitology.

[5]  M. Engstler,et al.  Endocytosis, membrane recycling and sorting of GPI‐anchored proteins: Trypanosoma brucei as a model system , 2004, Molecular microbiology.

[6]  M. Woolhouse,et al.  The origins of a new Trypanosoma brucei rhodesiense sleeping sickness outbreak in eastern Uganda , 2001, The Lancet.

[7]  Rick L. Tarleton,et al.  In Vitro and In Vivo High-Throughput Assays for the Testing of Anti-Trypanosoma cruzi Compounds , 2010, PLoS neglected tropical diseases.

[8]  Z. Zhou,et al.  Three-Dimensional Structure of the Trypanosome Flagellum Suggests that the Paraflagellar Rod Functions as a Biomechanical Spring , 2012, PloS one.

[9]  Christopher V. Rao,et al.  High-resolution, long-term characterization of bacterial motility using optical tweezers , 2009, Nature Methods.

[10]  Hongkai Wu,et al.  Single-cell assays. , 2011, Biomicrofluidics.

[11]  Elinore M Mercer,et al.  Microfluidic sorting of mammalian cells by optical force switching , 2005, Nature Biotechnology.

[12]  Beth Apsel,et al.  Discovery of Trypanocidal Compounds by Whole Cell HTS of Trypanosoma brucei , 2006, Chemical biology & drug design.

[13]  D. Marshall,et al.  Microfluidics for single cell analysis. , 2012, Current opinion in biotechnology.

[14]  I. Coppens,et al.  The uptake of the trypanocidal drug suramin in combination with low-density lipoproteins by Trypanosoma brucei and its possible mode of action. , 1993, Acta tropica.

[15]  Michael Unser,et al.  A pyramid approach to subpixel registration based on intensity , 1998, IEEE Trans. Image Process..

[16]  Stephan Herminghaus,et al.  Impact of Microscopic Motility on the Swimming Behavior of Parasites: Straighter Trypanosomes are More Directional , 2011, PLoS Comput. Biol..

[17]  E. Fèvre,et al.  Sleeping sickness in Uganda: a thin line between two fatal diseases , 2005, BMJ : British Medical Journal.

[18]  Michael P Barrett,et al.  Proline Metabolism in Procyclic Trypanosoma brucei Is Down-regulated in the Presence of Glucose* , 2005, Journal of Biological Chemistry.

[19]  A. Fairlamb.  Chemotherapy of human African trypanosomiasis: current and future prospects. , 2003, Trends in parasitology.

[20]  H. Andersson,et al.  Microfluidic devices for cellomics: a review , 2003 .

[21]  Michael P. Barrett,et al.  In Vivo Imaging of Trypanosome-Brain Interactions and Development of a Rapid Screening Test for Drugs against CNS Stage Trypanosomiasis , 2013, PLoS neglected tropical diseases.

[22]  Thomas Pfohl,et al.  Hierarchical self-assembly of actin in micro-confinements using microfluidics. , 2012, Biomicrofluidics.

[23]  Johannes E. Schindelin,et al.  Fiji: an open-source platform for biological-image analysis , 2012, Nature Methods.

[24]  Ling Yu,et al.  On-chip investigation of cell-drug interactions. , 2013, Advanced drug delivery reviews.

[25]  Mark C. Field,et al.  Receptor-mediated endocytosis for drug delivery in African trypanosomes: fulfilling Paul Ehrlich's vision of chemotherapy. , 2013, Trends in parasitology.

[26]  Jian Xu,et al.  Single-cell bioelectrical impedance platform for monitoring cellular response to drug treatment , 2011, Physical biology.

[27]  G. G. Stokes.  "J." , 1890, The New Yale Book of Quotations.

[28]  E. Fèvre,et al.  Identification of human-infective trypanosomes in animal reservoir of sleeping sickness in Uganda by means of serum-resistance-associated (SRA) gene , 2001, The Lancet.

[29]  E. Fèvre,et al.  Crisis, what crisis? Control of Rhodesian sleeping sickness. , 2006, Trends in parasitology.

[30]  Steven L Neale,et al.  Motile and non-motile sperm diagnostic manipulation using optoelectronic tweezers. , 2010, Lab on a chip.

[31]  S. Bodovitz,et al.  Single cell analysis: the new frontier in 'omics'. , 2010, Trends in biotechnology.

[32]  A. J. Nok.  Arsenicals (melarsoprol), pentamidine and suramin in the treatment of human African trypanosomiasis , 2003, Parasitology Research.

[33]  K. Matthews,et al.  The cell biology of Trypanosoma brucei differentiation. , 2007, Current opinion in microbiology.

[34]  David W. Gray,et al.  A Static-Cidal Assay for Trypanosoma brucei to Aid Hit Prioritisation for Progression into Drug Discovery Programmes , 2012, PLoS neglected tropical diseases.

[35]  K. Matthews,et al.  Molecular regulation of the life cycle of African trypanosomes. , 2004, Trends in parasitology.

[36]  Stephan Herminghaus,et al.  Hydrodynamic Flow-Mediated Protein Sorting on the Cell Surface of Trypanosomes , 2007, Cell.

[37]  J. Wiesner,et al.  Fosmidomycin for the treatment of malaria , 2003, Parasitology Research.

[38]  Kuo-Kang Liu,et al.  Optical tweezers for single cells , 2008, Journal of The Royal Society Interface.

[39]  W. Chiu,et al.  Structure of Trypanosoma brucei flagellum accounts for its bihelical motion , 2011, Proceedings of the National Academy of Sciences.

[40]  M. Girolami,et al.  The silicon trypanosome , 2010, Parasitology.

[41]  Nicole Rusk.  How parasites do it , 2005, Nature Methods.

[42]  M. Parsons,et al.  Active transport of 2-deoxy-D-glucose in Trypanosoma brucei procyclic forms. , 1990, Molecular and biochemical parasitology.

[43]  N. Perrimon,et al.  Droplet microfluidic technology for single-cell high-throughput screening , 2009, Proceedings of the National Academy of Sciences.

[44]  P. Büscher,et al.  Luminescent multiplex viability assay for Trypanosoma brucei gambiense , 2013, Parasites & Vectors.

[45]  J. Seed,et al.  INHIBITION OF HEXOSE AND GLYCEROL UTILIZATION BY 2-DEOXY-D-GLUCOSE IN TRYPANOSOMA GAMBIENSE AND TRYPANOSOMA RHODESIENSE. , 1965, Experimental parasitology.

[46]  E. Fèvre,et al.  Sleeping sickness: a tale of two diseases , 2001 .

[47]  C. Burri,et al.  Human African trypanosomiasis , 2010, The Lancet.

[48]  F. Richards.  LUNG BLOOD-FLOW IN MITRAL STENOSIS. , 1965, Lancet.

[49]  A. Manz,et al.  Revisiting lab-on-a-chip technology for drug discovery , 2012, Nature Reviews Drug Discovery.

[50]  Alex Groisman,et al.  A microfluidic chemostat for experiments with bacterial and yeast cells , 2005, Nature Methods.

[51]  M. Engstler,et al.  Optical trapping reveals propulsion forces, power generation and motility efficiency of the unicellular parasites Trypanosoma brucei brucei , 2014, Scientific Reports.