OpenSource Lab‐on‐a‐Chip Physiometer for Accelerated Zebrafish Embryo Biotests

Jin Akagi1, Chris J. Hall2, Kathryn E. Crosier2, Jonathan M. Cooper3, Philip S. Crosier2, Donald Wlodkowic1

1 The OpenTech Factory, School of Applied Sciences, RMIT University, Melbourne, 2 Department of Molecular Medicine and Pathology, School of Medical Sciences, University of Auckland, Auckland, 3 School of Engineering, University of Glasgow, Glasgow
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 9.44
DOI:  10.1002/0471142956.cy0944s67
Online Posting Date:  January, 2014
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Abstract

Zebrafish (Danio rerio) embryo assays have recently come into the spotlight as convenient experimental models in both biomedicine and ecotoxicology. As a small aquatic model organism, zebrafish embryo assays allow for rapid physiological, embryo‐, and genotoxic tests of drugs and environmental toxins that can be simply dissolved in water. This protocol describes prototyping and application of an innovative, miniaturized, and polymeric chip‐based device capable of immobilizing a large number of living fish embryos for real‐time and/or time‐lapse microscopic examination. The device provides a physical address designation to each embryo during analysis, continuous perfusion of medium, and post‐analysis specimen recovery. Miniaturized embryo array is a new concept of immobilization and real‐time drug perfusion of multiple individual and developing zebrafish embryos inside the mesofluidic device. The OpenSource device presented in this protocol is particularly suitable to perform accelerated fish embryo biotests in ecotoxicology and phenotype‐based pharmaceutical screening. Curr. Protoc. Cytom. 67:9.44.1‐9.44.16. © 2014 by John Wiley & Sons, Inc.

Keywords: Lab‐on‐a‐Chip; OpenSource; microfluidics; PDMS; zebrafish; biotest; bioassay; embryo; pharmacology; toxicology

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Zebrafish Embryo Culture and Embryo Preparation
  • Basic Protocol 2: Acrylic Master Mold Fabrication
  • Basic Protocol 3: PDMS Chip Fabrication and Assembly
  • Basic Protocol 4: Operation of the PDMS Chip‐Based Device
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Zebrafish Embryo Culture and Embryo Preparation

  Materials
  • Zebrafish embryos
  • E3 zebrafish medium
  • Assorted small dishes (5 to 10 ml; plasticware or glassware are both acceptable)
  • Magnifying glass or low‐magnifying stereomicroscope
  • Pasteur pipets

Basic Protocol 2: Acrylic Master Mold Fabrication

  Materials
  • Decon 90 detergent or similar
  • Transparent acrylic (PMMA; poly‐methyl methacrylate) sheets 2‐mm in thickness
  • Transparent acrylic (PMMA; poly‐methyl methacrylate) sheets 1.5‐mm in thickness
  • CNC laser cutter/engraver
  • CAD file
  • 25 × 75 × 3–mm steel metal bars
  • Mechanical G‐clamps
  • Oven

Basic Protocol 3: PDMS Chip Fabrication and Assembly

  Materials
  • Poly(dimethylsiloxane) elastomer (PDMS; Sylgard 184, Dow Corning)
  • Curing agent
  • 70% (v/v) ethanol
  • Oxygen plasma cleaner or atmospheric corona discharge unit
  • Degassing chamber capable of reaching 40 Torr
  • Master mold (see protocol 2)
  • Glass petri dish capable of holding 25 × 75–mm molds
  • Oven
  • Set of scalpels
  • Biopsy punch hole
  • 1/16‐in. polyurethane tubing (Cole‐Parmer) with i.d. of 1.5 mm

Basic Protocol 4: Operation of the PDMS Chip‐Based Device

  Materials
  • 70% (v/v) ethanol
  • E3 medium (see recipe)
  • Zebrafish embryos
  • Drug or toxin to stimulate the embryos
  • PDMS device assembled as described in protocol 3
  • Peristaltic pump with a flow rate adjustable between 0.1 to 2 ml/min
  • Small vessels for preparing embryos
  • Microscope equipped with a time‐lapse camera and heated stage
  • Small water bath or dry block heater
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Figures

Videos

Literature Cited

Literature Cited
  Akagi, J., Khoshmanesh, K., Evans, B., Hall, C.J., Crosier, K.E., Cooper, J.M., Crosier, P.S., and Wlodkowic, D. 2012a. Miniaturized embryo array for automated trapping, immobilization and microperfusion of zebrafish embryos. PLoS One 7:e36630.
  Akagi, J., Khoshmanesh, K., Hall, C.J., Cooper, J.M., Crosier, K.E., Crosier, P.S., and Wlodkowic, D. 2012b. Fish on chips: Microfluidic living embryo array for accelerated in vivo angiogenesis assays. Sensors Actuators B 189:11‐20.
  Buchanan, C.M., Shih, J.H., Astin, J.W., Rewcastle, G.W., Flanagan, J.U., Crosier, P.S., and Shepherd, P.R. 2012. DMXAA (Vadimezan, ASA404) is a multi‐kinase inhibitor targeting VEGFR2 in particular. Clin. Sci. 122:449‐457.
  Chakraborty, C., Hsu, C.H., Wen, Z.H., Lin, C.S., and Agoramoorthy, G. 2009. Zebrafish: A complete animal model for in vivo drug discovery and development. Curr. Drug Metab. 10:116‐124.
  Chiu, L.L., Cunningham, L.L., Raible, D.W., Rubel, E.W., and Ou, H.C. 2008. Using the zebrafish lateral line to screen for ototoxicity. Jaro‐J Assoc. Res. Oto. 9:178‐190.
  Eimon, P.M. and Rubinstein, A.L. 2009. The use of in vivo zebrafish assays in drug toxicity screening. Exp. Opin. Drug Metab. Toxicol. 5:393‐401.
  Evensen, L., Link, W., and Lorens, J.B. 2010. Imaged‐based high‐throughput screening for anti‐angiogenic drug discovery. Curr. Pharm. Design 16:3958‐3963.
  Huang, X.Q., Nguyen, A.T., Li, Z., Emelyanov, A., Parinov, S., and Gong, Z.Y. 2011. One step forward: The use of transgenic zebrafish tumor model in drug screens. Birth Defects Res. C 93:173‐181.
  Kamei, M., Isogai, S., Pan, W.P., and Weinstein, B.M. 2010. Imaging blood vessels in the zebrafish. Methods Cell Biol. 100:27‐54.
  Khoshmanesh, K., Akagi, J., Hall, C.J., Crosier, K.E., Crosier, P.S., Cooper, J.M., and Wlodkowic, D. 2012. New rationale for large metazoan embryo manipulations on chip‐based devices. Biomicrofluidics 6:24102‐2410214.
  Kidd, K.R. and Weinstein, B.M. 2003. Fishing for novel angiogenic therapies. Br. J. Pharmacol. 140:585‐594.
  Kusik, B.W., Carvan, M.J.3rd, and Udvadia, A.J. 2008. Detection of mercury in aquatic environments using EPRE reporter zebrafish. Mar. Biotechnol. 10:750‐757.
  Kuster, E. and Altenburger, R. 2008. Oxygen decline in biotesting of environmental samples—is there a need for consideration in the acute zebrafish embryo assay? Environ. Toxicol. 23:745‐750.
  Lammer, E., Carr, G.J., Wendler, K., Rawlings, J.M., Belanger, S.E., and Braunbeck, T. 2009a. Is the fish embryo toxicity test (FET) with the zebrafish (Danio rerio) a potential alternative for the fish acute toxicity test? Comp. Biochem. Physiol. C 149:196‐209.
  Lammer, E., Kamp, H.G., Hisgen, V., Koch, M., Reinhard, D., Salinas, E.R., Wendler, K., Zok, S., and Braunbeck, T. 2009b. Development of a flow‐through system for the fish embryo toxicity test (FET) with the zebrafish (Danio rerio). Toxicol. In Vitro 23:1436‐1442.
  Lawson, N.D. and Weinstein, B.M. 2002. In vivo imaging of embryonic vascular development using transgenic zebrafish. Develop. Biol. 248:307‐318.
  Lieschke, G.J. and Currie, P.D. 2007. Animal models of human disease: Zebrafish swim into view. Nat. Rev. Genet. 8:353‐367.
  Okuda, K.S., Astin, J.W., Misa, J.P., Flores, M.V., Crosier, K.E., and Crosier, P.S. 2012. lyve1 expression reveals novel lymphatic vessels and new mechanisms for lymphatic vessel development in zebrafish. Development 139:2381‐2391.
  Pardo‐Martin, C., Chang, T.Y., Koo, B.K., Gilleland, C.L., Wasserman, S.C., and Yanik, M.F. 2010. High‐throughput in vivo vertebrate screening. Nat. Methods 7:634‐636.
  Parng, C. 2005. In vivo zebrafish assays for toxicity testing. Curr. Opin. Drug Discov. 8:100‐106.
  Parng, C., Seng, W.L., Semino, C., and McGrath, P. 2002. Zebrafish: A preclinical model for drug screening. Assay Drug Dev. Technol. 1:41‐48.
  Sia, S.K. and Whitesides, G.M. 2003. Microfluidic devices fabricated in poly(dimethylsiloxane) for biological studies. Electrophoresis 24:3563‐3576.
  Tan, W.H. and Takeuchi, S. 2007. A trap‐and‐release integrated microfluidic system for dynamic microarray applications. Proc. Natl. Acad. Sci. U.S.A. 104:1146‐1151.
  Tan, W.H. and Takeuchi, S. 2008. Dynamic microarray system with gentle retrieval mechanism for cell‐encapsulating hydrogel beads. Lab Chip 8:259‐266.
  Weigt, S., Huebler, N., Strecker, R., Braunbeck, T., and Broschard, T.H. 2011. Zebrafish (Danio rerio) embryos as a model for testing proteratogens. Toxicology 281:25‐36.
  Wlodkowic, D., Khoshmanesh, K., Akagi, J., Williams, D.E., and Cooper, J.M. 2011. Wormometry‐on‐a‐chip: Innovative technologies for in situ analysis of small multicellular organisms. Cytometry A 79:799‐813.
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