An ex-ovo Chicken Embryo Culture System Suitable for Imaging and Microsurgery Applications

Summary

In this article, we present a simple methodology to enable long-term ex-ovo avian embryo culture. This technique is ideal for longitudinal experimentation requiring complete optical accessibility and/or sterile transportation in avian embryos.

Abstract

Understanding the relationships between genetic and microenvironmental factors that drive normal and malformed embryonic development is fundamental for discovering new therapeutic strategies. Advancements in imaging technology have enabled quantitative investigation of the organization and maturing of the body plan, but later stage embryonic morphogenesis is less clear. Chicken embryos are an attractive vertebrate animal model system for this application because of its ease of culture and surgical manipulation. Early embryos can be cultured for a short time on filter paper rings, which enables complete optical access for cell patterning and fate studies^1,2. Studying advanced developmental processes such as cardiac morphogenesis are traditionally performed through a window of the eggshell^3-5, but this technique limits optical access due to window size. We previously developed a simple method to culture whole embryos ex-ovo on hexagonal weigh boats for up to 10 days, which enabled high resolution imaging via ultrasonography^6,7. These cultures were difficult to transport, limiting the types of imaging tools available for live experiments. We here present an improved shell-less culture system with a cost-effective, portable environmental chamber. Eggs were cracked onto a hammock created by a polyurethane membrane (cling wrap) affixed circumferentially to a plastic cup partially filled with sterile water. The dimensions of the circumference and depth of the hammock were both critical to maintain surface tension, while the mechanics of the hammock and water beneath helped dampen vibrations induced by transportation. A small footprint circulating water bath was also developed to enable continuous temperature control during experimentation. We demonstrate the ability to culture embryos in this way for at least 14 days without morphogenic defect or delay and employ this system in several microsurgical and imaging applications.

Protocol

1. Ex-ovo Culture Protocol:

1. Incubate fertilized chicken eggs
   1. Fertilized chicken eggs can be stored at 13°C up to 5 days before incubation without initiating development. A red wine cooler can maintain this temperature.
   2. Incubate eggs blunt side up in a 60% constant humidity incubator with continuous rocking at 37.5°C for 72 hours.
2. Prepare hammocks (Figure 1a)
   1. Fill ¾ of 9 oz plastic cups with warm sterile water. A regular 9 oz cup has a top diameter of 8 cm. We found that this size was most suitable for long time culture application prese....

Discussion

Optical access and experimentation in avian embryos is challenging due to constraints of the egg shell. Windowing significantly limits the number of microvessels accessible for injection and microsurgical approaches⁸. As a result only early embryos can be manipulated and continuous observation is not possible. Early ex-ovo cultures using Petri dishes were of limited use because of inadequate control of the surface tension on the embryo prevented long term culture⁹. We recently improved .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Fertile white Leghorn chicken eggs
Model GB1, Avery Incubators, Hugo CO
Saran Wrap
Kimwipes	Kimberly-Clark Corporation
Rubber bands
Warm sterile water
9 oz plastic cup
100 mm diameter Petri dish
1602N thermal air	GQF Manufacturing
Fluorescein-conjugated dextran (2 MDa, 1% w/v in phosphate buffered saline)	Sigma-Aldrich
Microforge	Glassworx, Inc, St Louis MO
Glass capillary tubes (0.75 mm ID)
Micromanipulator	World Precision Instruments, Inc.	Model M3301L
Fluorescent microscopy	Carl Zeiss, Inc.	Z20
Fine 55-forceps	World Precision Instruments, Inc.
10-0 nylon surgical suture	Ethicon Inc.
Tubing	VWR international		1mm OD
3 mL syringe	BD Biosciences
200 μL pipetter and pipette tips	VWR international