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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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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Basic Protocol 1: Zebrafish Embryo Culture and Embryo Preparation

  • 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

  • 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

  • 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

  • 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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Literature Cited

Literature Cited
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