1Department of Biomedical Engineering, Cornell University, 2Current Address: Mechanical Engineering Department, Dogus University
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Yalcin, H. C., Shekhar, A., Rane, A. A., Butcher, J. T. An ex-ovo Chicken Embryo Culture System Suitable for Imaging and Microsurgery Applications. J. Vis. Exp. (44), e2154, doi:10.3791/2154 (2010).
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 studies1,2. Studying advanced developmental processes such as cardiac morphogenesis are traditionally performed through a window of the eggshell3-5, 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 ultrasonography6,7. 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.
1. Ex-ovo Culture Protocol:
Accessory equipment for outside incubator imaging/ manipulation:
A circulating water bath was built in house to maintain incubation temperatures while imaging or manipulation (Figure 3). A water bath that can fit the cup is connected via appropriate size tubing to a water reservoir. A resistance heater heats the water in the reservoir which is regulated manually by following the water temperature. A water pump circulates the water. Pictoral representation depicts imaging ease via ultrasongraphy/florescent microscopy (Figure 3).
Figure 1. Preparation of hammock and transfer of chicken embryos to hammocks.
Figure 2. Representative results - different stage chicken embryos grown via ex ovo culture technique. Embryos from HH 17 onwards can be grown via this technique.
Figure 3. Schematic and representative pictures of the water circulation system used in conjunction with the ex-ovo culture system to keep embryos at ideal development temperature.
2. Selected Applications:
This embryo culture is suitable for microinjection applications where solutions need to be injected to vasculature for contrast based imaging (ie. fluorescent microscopy, micro-computed topography etc.) (Figure 4). Below is the microinjection protocol:
Figure 4: Microinjection of a fluorescent dye into the vasculature of an ex-ovo cultured chick embryo.
II. Microsurgical Procedure - Left Atrial Ligation
This ex-ovo culture setup is ideal for performing surgical applications to chick embryos since it provides full accessibility to the embryo. Below is the protocol for an example technique, left atrial ligation (LAL) (Figure 5). The procedure is performed approximately 24 hours into the culture period (HH24):
Figure 5: A control and a left atrial ligated chick embryo. Arrow shows the left atria, which is about 75% smaller in the ligated embryo.
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 this technique using a hexagonal weigh boat that maintains acceptable surface tenstion7, but these cultures were difficult to transport. The technique presented here solves this problem and significantly expands the type of experimental methods and imaging techniques that can be employed including microinjection and surgical procedures. As research focuses on understanding later morphogenic events, this technique will enable monitoring and modulation of development events that are currently impossible to experimentally study continuously.
No conflicts of interest declared.
|Fertile white Leghorn chicken eggs|
|Model GB1, Avery Incubators, Hugo CO|
|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 ÁL pipetter and pipette tips||VWR international|