A 3-dimensional (3D)-printed Template for High Throughput Zebrafish Embryo Arraying

Summary

Here, we present a protocol to design and fabricate a zebrafish embryo arraying template, followed by a detailed procedure on the use of such template for high throughput zebrafish embryo arraying into a 96-well plate.

Abstract

The zebrafish is a globally recognized fresh water organism frequently used in developmental biology, environmental toxicology, and human disease related research fields. Thanks to its unique features, including large fecundity, embryo translucency, rapid and simultaneous development, etc., zebrafish embryos are often used for large scale toxicity assessment of chemicals and drug/compound screening. A typical screening procedure involves adult zebrafish spawning, embryos selection, and arraying the embryos into multi-well plates. From there, embryos are subjected to exposure and the toxicity of chemical, or the effectiveness of the drugs/compounds can be evaluated relatively quickly based on phenotypic observations. Among these processes, embryos arraying is one of the most time-consuming and labor-intensive steps that limits the throughput level. In this protocol, we present an innovative approach that makes use of a 3D-printed arraying template coupled with vacuum manipulation to speed up this laborious step. The protocol herein describes the overall design of the arraying template, a detailed experimental setup and step-by-step procedure, followed by representative results. When implemented, this approach should prove beneficial in a variety of research applications using zebrafish embryos as testing subjects.

Introduction

As a popular model organism, zebrafish is widely used in the fields of medicine and toxicology^1,2,3,4. Compared to in vitro platforms, zebrafish offer much greater biological complexities that one or two cell types could not offer. Besides being a whole organism model, the zebrafish's large fecundity, rapid and simultaneous embryonic development, and high organ translucency have given this model unique advantages to be used for large scale toxicity or drugs/compound screening⁵. The hundreds of embryos produced by one pair of adult zebrafish each week surpass any other whole animal models and have made it suitable for high throughput screening.

A typical screening procedure using zebrafish involves a significant amount of manual work, such as adult zebrafish spawning, embryo selection, and arraying embryos into suitable containers where they are subjected to exposure through water immersion. The development of the embryos is monitored and observable endpoints such as mortality, hatchability and abnormality are often evaluated manually and used as the preliminary identifications of the toxicity of chemicals or indications of the effectiveness of drugs or compounds. To speed up the screening procedure, approaches such as automated imaging and computer-assisted image analysis have been explored previously. For example, microscopes with high content imaging capabilities have been adapted to perform automated bright-field or fluorescence imaging on zebrafish embryos at various developmental stages from 96/384 well plates⁶. Microfluidic devices coupled with microscopes were used to position zebrafish larvae through current manipulation for imaging of brain neurons⁷. These approaches could significantly improve the efficiency of image acquisitions compared to traditional manual operation. Moreover, with large number of images being generated, image analysis tools have also been developed to speed up the data processing, as demonstrated by Liu et al. and Tu et al.^8,9.

As the throughput level of imaging and image analysis increases, it became clear that the rate-limiting step for screening lies in the process of preparing zebrafish embryos for exposure, which typically means arraying them into 96- or 384-well plates. To solve this bottleneck step, vision-guided robotics were developed by Mandrell et al.¹⁰ and us¹¹ previously to replace manual handling but the instruments were rather sophisticated and there is a deep learning curve to implement such techniques. Therefore, to provide an easy-to-use approach becomes one important factor to further improve the throughput level of zebrafish screening and is the main objective of this work.

In this work, we designed and fabricated an embryo arraying template by 3D printing. Such an arraying template was designed to entrap zebrafish embryo into wells that fit with a standard 96-well plate. Instead of selecting embryos and arraying them into individual well one by one, one could perform embryo entrapment and array all 96 embryos into a multiwall plate at once. Using this template and the following protocol, one could significantly increase the efficiency of arraying embryos into multiwall plates, which would in term boost the screening capacity at least tenfold, compared to manual operation. The protocol described below includes an overall design for the arraying template, zebrafish spawning, embryo collection, and arraying. Figure 1 shows the overall design of the arraying template. Figure 2 shows an overview of the step-by-step protocol on using the template described in Parts 3 and 4.

Protocol

1. Design and Fabrication of a Zebrafish Embryos Arraying Template

1. Design the arraying template with a 12 by 8, 96-well layout that fits a standard 96-well plate. Use the dimensions listed in Figure 1A for the upper embryo entrapment chamber (see also the Supplemental File).
   1. Use the dimensions shown in Figure 1B and 1D for the entrapment well.
   2. Use the dimensions in Figure 1C for the bottom vacuum chamber.
   3. Use the dimensions in Figure 1B for the air in/outlet.
2. Use a 3D-printer (with 0.1 mm precision) to print the template; see Table of Materials for recommended resin to be used for printing.
   NOTE: 3D printers with a 0.1 mm precision are recommended for fabrication of the arraying template (see Table of Materials). The suggested color for the surface of the template is dark grey or black.

2. Zebrafish Embryo Spawning

1. Place two pairs of male and female fish per mating box one day prior to spawning. Separate males and females by a clear plastic divider.
2. Take off the dividers in the morning to mix male and female fish.
3. Remove the male and female fish and collect zebrafish embryos using a fine-mesh strainer. Wash the embryos with 250 mL egg water (see Table of Materials).
4. Transfer the collected embryos to petri dishes (90 mm in diameter) with Holtfreter's solution (see Table of Materials) and remove dead and unfertilized embryos using a stereomicroscope.
5. Place the embryos in a 28.5 °C incubator. At 4 h post fertilization (hpf), observe the embryos and remove any dead and unhealthy embryos. The embryos are now ready for the next step.

3. Preparation of Arraying Template

1. Wash the template 2–3 times with 500 mL deionized water and put it into the drying oven (45 °C) for 5 min.
2. Tape the bottom chamber with a piece of sealing film (Figure 2, Step 1).
3. Connect a vacuum pump through the air outlet at the bottom of the template.
   NOTE: The recommended max vacuum for the vacuum pump is 0.1 Mpa. Be aware of the strength of the vacuum used. If the negative pressure is too strong, cut a cross-shaped hole on the sealing film to lower the pressure.

4. Arraying Zebrafish Embryos into a 96-well plate

1. Using a plastic transfer pipette, place approximately 150 embryos into the template, as demonstrated in Figure 2, Step 2.
2. Connect the vacuum pump to the air outlet to generate negative pressure in the chamber sealed by the sealing film in step 3.3.
3. Shake the entire template horizontally until each well has one embryo entrapped (Figure 2, Step 3).
   NOTE: If the Holtfreter's Solution dries up before the embryos are trapped in each well, add additional Holtfreter's Solution in the entrapment chamber and repeat this step.
4. Discard extra Holtfreter's Solution and embryos that are not entrapped in the wells (Figure 2, Step 4).
5. Turn off and disconnect the vacuum pump.
6. Place a standard 96-well plate upside down against the template (Figure 2, Step 5) and rotate both at the same time (Figure 2, Step 6).
7. Tap the bottom of the template or connect the air outlet to a compressed gas dusting can to transfer all trapped embryos from the template to the 96-well plate (Figure 2, Step 6).
8. Repeat step 4.1 to 4.8 to prepare additional multi-well plates.
9. Remove the sealing film and wash the template 3 times from top to bottom with 500 mL deionized water for future use.
   NOTE: Do not use any organic solvents, like ethanol, to clean the template.

Results

Figure 3 shows a typical 3D-printed arraying template. This template uses photosensitive resin as raw material and was made by a 3D printer; a layer of black paint was applied to provide a better contrast to the color of embryos. The position of 96 wells (12 by 8) was designed to fit with a standard 96-well plate. Similarly, a 384 (24 by 16) well template could also be designed and fabricated using the same method. The upside chamber was slightly bigger than ...

Discussion

There are two critical steps in this protocol that require close attention for a successful implementation of 3D-printed template for arraying zebrafish embryos.

The most important factor on the design of the arraying template is the entrapment well. To makes sure there is only one embryo trapped in each well, one should pay close attention to the diameter and the depth of the entrapment well, and the diameter of the through hole. The recommended diameter is within 1.5 to 2 times of the diamet...

Materials

Name	Company	Catalog Number	Comments
Zebrafish Facility	Shanghai Haisheng Biotech Co., Ltd.	Z-A-S5
Mating box	Shanghai Haisheng Biotech Co., Ltd.
Wash Bottle, 500 ml	Sangon Biotech	F505001-0001
Sodium chloride	Vetec	V900058-500G
Potassium Chloride	Sinopharm Chemical Reagent Co.,Ltd	10016318
Calcium chloride	Sinopharm Chemical Reagent Co.,Ltd	20011160
Sodium bicarbonate	Vetec	v900182-500G
Methylene Blue Hydrate	TCI	M0501
Hydrochloric acid	Sinopharm Chemical Reagent Co.,Ltd	10011008
Sea Salts	Instant Ocean	SS15-10
Pipetter	Fisherbrand	13-675M
Controlled Drop Pasteur Pipet	Fisherbrand	13-678-30
Microscope	OLYMPUS	SZ61
Biochemical incubator	Shanghai Yiheng Scientific Instrument Co., Ltd.	LRH-250
3D printer	UnionTech	Lite600
Photosensitive resin	UnionTech	UTR9000
Vacuum pump	Shanghai Yukang Scientific Instrument Co., Ltd.	SHB-IIIA
Adhesive PCR Plate Seals	Solarbio	YA0245
96 well plate	Costar	3599
Multi 8-channel pipette 30 - 300 μl	Eppendorf	3122000.051
Compressed Gas Duster	Shanghai Zhantu Chemical Co., Ltd.	ST1005
DI Water	Thermo	GenPure Pro UV/UF
Drying oven	Shanghai Yiheng Scientific Instrument Co., Ltd.	BPG-9106A
System water			Water out of the facility’s water system
Egg water			Dilute 60mg “Instant Ocean” sea salts and 0.25 mg/L methylene blue in 1 L DI water
Holtfreter’s solution			Dissolve 7.0 g Sodium chloride (NaCl), 0.4 g Sodium bicarbonate (NaHCO3), 0.1 g Potassium Chloride (KCl), 0.235 g Calcium chloride (CaCl2.2H2O) in 1.9 L DI water. Adjust pH to 7 using HCl and adjust volume to 2 L using Di water