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, null, 2 Department of Molecular Medicine and Pathology, School of Medical Sciences, University of Auckland, Auckland, null, 3 School of Engineering, University of Glasgow, Glasgow, null
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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  •   FigureFigure 9.44.1 Hydrodynamic embryo‐trapping array. (A) 2D CAD drawing outlining the geometry of the device for trapping and immobilization of large metazoan embryos. Note the array of 48 traps interconnected with an array of 48 cross‐flow suction channels. Blue arrows denote the direction of fluid flow. R—denotes the numbering of consecutive trapping rows; T—denotes the numbering of consecutive traps; (B) Photograph of the assembled polymeric device. Left panel depicts a magnified section of the chip with a single trap holding an immobilized zebrafish embryo.
  •   FigureFigure 9.44.2 Workflow of rapid prototyping technique. (A) Laser cutting of a PMMA negative relief pattern, (B) dust particles removal, and (C) ‐PMMA thermal bonding. C‐clamps are used to apply a uniform mechanical force during the bonding process and (D) PDMS replica molding. Note that PDMS does not bind electrostatically or covalently to PMMA, allowing rapid removal without the need for releasing agents.
  •   FigureFigure 9.44.3 Operation and fluidic network connecting the device with external actuators. (A) Schematic diagram of the fluidic connections required to operate the chip‐based device under open‐loop (blue/dashed lines) and closed‐loop (black/solid lines) perfusion and the hardware components actuating the microfluidic device. (B) Photograph depicting a sample chip‐based device operating in a closed‐loop perfusion and interconnected with time‐lapse stereomicroscope, peristaltic pump, and a sample drug reservoir inside a water bath.
  •   FigureFigure 9.44.4 Hydrodynamic embryo‐trapping principles inside a miniaturized chip‐based device. (A) Streamlines of fluid colored by flow velocity (m/sec) across the inlet section of the device at the vertical middle plane when perfused at a flow rate of 1 ml/min. (B) A 3D cartoon showing the embryo‐trapping principles: (1) the embryo is aspirated from the storage vessel and injected into the main channel, (2) hydrodynamic forces guide the embryo into the trap, (3 and 4) the next embryo is introduced and rolls on the previous one towards the next available trap, (5 and 6) the process is repeated until all of the traps are filled with embryos, while the hydrodynamic forces keep embryos securely docked for the duration of the experiments. (C) Time‐lapse imaging of the trapping process inside the chip‐based device. Numbers depict sequential rows containing miniaturized hydrodynamic trapping modules (refer to Fig. for a detailed CAD schematics). Note that due to the hydrodynamic design of the chip, initial trapping might not be in sequence and embryos tend to skip the traps in the first row (dashed rectangle). Moreover, initial trapping will be slow and will rapidly accelerate as the number of trapped embryos increase. This is because trapped embryos act as plugs that increase the flow resistance across the occupied trap and redirect more flow to the subsequent traps in each row. After filling the first two rows, the trapping process becomes highly ordered and sequential. The blue arrow depicts the direction of the fluid flow.
  •   FigureFigure 9.44.5 Time‐resolved imaging of zebrafish embryo development under continuous microperfusion. (A) Time‐lapse images of developing zebrafish embryos collected every 24 hr. Embryos were loaded on a chip at the volumetric flow rate of 2 ml/min. Subsequently, the chip was perfused at a rate of 0.4 ml/min for up to 48 hr; (B) Transgenic zebrafish line fli1a:EGFP embryos at 16 hpf were loaded, immobilized, and continuously perfused on a chip with E3 medium containing vehicle control (DMSO). Fluorescent and bright‐field images were acquired at 0, 24, and 48 hr intervals. The optical transparency of embryos coupled with hydrodynamic immobilization on a chip array allowed for convenient microscopic visualization of characteristic patterns of intersegmental vessels (ISV, white arrows).


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