The authors would like to thank Gregory Haller (Mount Saint Vincent University) for his preliminary work in the development of the protocol. The authors would also like to thank Nicholas Jones (Mount Saint Vincent University) for his technical expertise and assistance with the filming and production of the audio/visual portion of the manuscript. Daniel Andrews was supported by funding from MSVU and the Natural Sciences and Engineering Research Council of Canada (NSERC) via an Undergraduate Student Research Award. Tamara A. Franz-Odendaal is supported by an NSERC Discovery Grant.

NOTE: For embryo stages, utilize the Hamburger and Hamilton12 (HH) staging table.
1. Embryo Incubation
<ol>
	<li>Incubate fertilized chicken eggs in a sterile, temperature-controlled incubator at 37 &#176;C &#177; 1 &#176;C and ~40% humidity.</li>
	<li>Turn the eggs 1x per day and allow them to incubate to HH34 (8 days post-fertilization).</li>
</ol>
2. Preparation and Sterilization of the Materials
<ol>
	<li>For 12 embryos, autoclave 2 L of distilled water, 1 L of 0.85% chick saline, 12 glass pipettes, 1 box of paper tissue, 24 2.5 cm x 2.5 cm squares of semi-porous filter paper, and 24 2.5 cm x 2.5 cm squares of steel wire mesh with the edges curved downwards. To sterilize the materials, autoclave them at 121 &#176;C for at least 15 min. 
	NOTE: The autoclaving of the materials can be done in advance.</li>
	<li>Sterilize the incubator with 70% ethanol by wiping the sides, shelves, and door. Place 2 plastic containers containing autoclaved distilled water inside the incubator to maintain humidity. Prewarm the culture incubator to 37 &#176;C &#177; 1 &#176;C and ~40% humidity. Ensure the incubator is completely dark; if the door is transparent, cover it with tinfoil.</li>
	<li>Prewarm 100 mL of nutrient medium and 1 mL of penicillin by placing them in the culture incubator.</li>
	<li>Cover the workspace with a paper towel and sterilize the bench by spraying it with 70% ethanol. Sterilize 2 pairs of forceps, dissection scissors, a razor blade, and a plastic spoon with 70% ethanol in a similar manner.</li>
	<li>Place 12 sterile 100 mm Petri dishes and 24 sterile 35 mm Petri dishes on the bench for use. Ensure the dishes remain in a sterile bag until use.</li>
</ol>
3. Embryo Preparation
NOTE: From this point onwards, wear a protective dust face mask to avoid contaminating the cultures. A bacterial or fungal infection will ruin the culture and there is a risk of it spreading quickly to all cultures in the incubator.
<ol>
	<li>Crack open the egg and transfer the embryo to a 100 mm Petri dish containing 0.85% chick saline. Stage the embryo according to the anatomical features described in the Hamburger and Hamilton staging guidelines12 and confirm the embryo is at HH34.</li>
	<li>Cut the neck and then bisect the head of the embryo at the midline using a sterilized razor blade. Using sterile forceps, remove the brain. Leave the beak intact.</li>
</ol>
4. Culture Setup
<ol>
	<li>Place 2 of the 35 mm Petri dishes inside one 100 mm Petri dish.</li>
	<li>Place a steel wire mesh inside each 35 mm Petri dish.</li>
	<li>Using forceps, transfer 1 of the bisected heads to a piece of 2.5 cm x 2.5 cm semi-porous filter paper with the eye facing up. Ensure the tissue is secure and does not slip off by tilting it slightly.</li>
	<li>Using forceps, carefully place the eye tissue and the filter paper on top of the steel wire mesh in 1 of the 35 mm Petri dishes, creating a raised stage with the eye tissue on top.</li>
	<li>Repeat steps 4.3 and 4.4 with the other half head.</li>
	<li>Using a sterile glass pipette, carefully add nutrient medium directly into each 35 mm Petri dish until it reaches the level of the filter paper. Do not submerge the half head in the nutrient medium.</li>
	<li>Add 50 &#181;L of 10,000 U/mL penicillin-streptomycin to the nutrient medium in each Petri dish.</li>
	<li>Fold a piece of tissue paper into a small square and place it inside the 100 mm Petri dish. Moisten it with autoclaved distilled water.</li>
	<li>Place the culture dish into the sterile, dark incubator at 37 &#176;C &#177; 1 &#176;C and ~40% humidity for up to 4 days.</li>
	<li>Repeat steps 3 and 4 for each embryo. 
	NOTE: The number of embryos needed will depend on the specific experiment; here, we describe the protocol for 12 embryos.</li>
</ol>
5. Culture Maintenance
<ol>
	<li>Once per day, top up the plastic containers in the incubator with fresh, autoclaved distilled water.</li>
	<li>Daily check all cultures for bacterial or fungal infections. Cultures that have disintegrated or in which the medium has changed color are infected. Discard all cultures that are infected.</li>
</ol>
6. Fixation
<ol>
	<li>Following culturing, remove all dishes from the incubator.</li>
	<li>Using a pair of forceps, gently remove the eye from the filter paper, taking care not to tear the tissue.</li>
	<li>Fix the eye tissue in 4% paraformaldehyde in 1x phosphate-buffered saline overnight at 4 &#176;C or in 10% neutral-buffered formalin overnight at room temperature.</li>
	<li>Store the tissue at 4 &#176;C in 1x phosphate-buffered saline.</li>
</ol>

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	<li>Franz-Odendaal, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Towards+understanding+the+development+of+scleral+ossicles+in+the+chicken,+Gallus+gallus.">Towards understanding the development of scleral ossicles in the chicken, Gallus gallus.</a> Developmental Dynamics. 237 (11), 3240-3251 (2008).</li><li>Scholey, J. 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I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+organ+culture+of+embryonic+chick+femora:+a+system+for+investigating+bone+and+cartilage+formation+at+an+intermediate+level+of+organization.">Long-term organ culture of embryonic chick femora: a system for investigating bone and cartilage formation at an intermediate level of organization.</a> Journal of Bone and Mineral Research. 5 (1), 85-100 (1990).</li><li>Jung, H. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Local+inhibitory+action+of+BMPs+and+their+relationships+with+activators+in+feather+formation:+implications+for+periodic+patterning.">Local inhibitory action of BMPs and their relationships with activators in feather formation: implications for periodic patterning.</a> Developmental Biology. 196 (1), 11-23 (1998).</li><li>McKinnell, I. W., Turmaine, M., Patel, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sonic+Hedgehog+functions+by+localizing+the+region+of+proliferation+in+early+developing+feather+buds.">Sonic Hedgehog functions by localizing the region of proliferation in early developing feather buds.</a> Developmental Biology. 272 (1), 76-88 (2004).</li><li>Smith, E. L., Kanczler, J. M., Oreffo, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+take+on+an+old+story:+chick+limb+organ+culture+for+skeletal+niche+development+and+regenerative+medicine+evaluation.">A new take on an old story: chick limb organ culture for skeletal niche development and regenerative medicine evaluation.</a> European Cells &amp; Materials. 26, 91-106 (2013).</li><li>Sato, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+Vitro+Spermatogenesis+in+Explanted+Adult+Mouse+Testis+Tissues.">In Vitro Spermatogenesis in Explanted Adult Mouse Testis Tissues.</a> PLoS One. 10 (6), e0130171 (2015).</li><li>Ochoa-Villarreal, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant+cell+culture+strategies+for+the+production+of+natural+products.">Plant cell culture strategies for the production of natural products.</a> BMB Reports. 49 (3), 149-158 (2016).</li><li>Hamburger, V., Hamilton, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+series+of+normal+stages+in+the+development+of+the+chick+embryo.">A series of normal stages in the development of the chick embryo.</a> Journal of Morphology. 88 (1), 49-92 (1951).</li><li>Yu, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+developmental+biology+of+feather+follicles.">The developmental biology of feather follicles.</a> The International Journal of Developmental Biology. 48, 181-191 (2004).</li><li>Jabalee, J., Hillier, S., Franz-Odendaal, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+investigation+of+cellular+dynamics+during+the+development+of+intramembranous+bones:+the+scleral+ossicles.">An investigation of cellular dynamics during the development of intramembranous bones: the scleral ossicles.</a> Journal of Anatomy. 223 (4), 311-320 (2013).</li><li>Trowell, O. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+culture+of+mature+organs+in+a+synthetic+medium.">The culture of mature organs in a synthetic medium.</a> Experimental Cell Research. 16 (1), 118-147 (1959).</li><li>Pinto, C. B., Hall, B. 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The authors have nothing to disclose.

This protocol makes use of established tissue culturing techniques to achieve the growth of a chicken eye from embryonic day 8 (HH34) in vitro for 4 days. This grid-culturing method was originally described by Trowell15. We optimized a protocol from Pinto and Hall&#39;s 1991 study16 utilizing a semi-porous membrane with the grid to study inductive signals between separated tissue layers of the embryonic chicken eye15. Using this method, Roac...

The embryonic chicken (Gallus gallus) is an excellent model organism commonly used in the field of biology. Its incubation period is roughly 21 days and many eggs can be incubated simultaneously, making experimentation quick and efficient. Perhaps most importantly, the embryo is also easily manipulated, enabling the extensive study of key developmental processes and of the genes and proteins that drive these processes.
The embryonic chicken eye is a complex organ that develops via the interaction of a number of different tissues similar to many other body systems. This method enables the study of the development of these tissues, particularly at advanced stages of development. For example, the multi-layered retina may be of particular interest to those studying the development of the nervous system. Alternative methods that enable the study of other eye tissues such as the cornea, the vitreal body, the lens, the sclera, and the eyelids are of benefit to researchers. The chicken embryonic eye also contains a series of flat bones, the scleral ossicles, which can be used as a model for the study of intramembranous bone induction and ossification in vertebrates1.
Currently, there are a number of methods used to study embryonic development. Microinjections of inhibitory antibodies or other inhibitory molecules2,3, surgically implanted microbeads soaked in inhibitor4, and electroporation5 are all methods that can be used to downregulate genes or proteins of interest in an embryo. Similar methods are used to upregulate proteins. These methods are not without their limitations. For example, when using chemicals to alter the embryonic development, the lethal effects on the embryo must be evaluated, and this limits the use of the aforementioned methods to localized sites of application at doses low enough to ensure the survivability of the embryo.
In vitro tissue&#160;culturing has been used in a wide range of organisms to study development and can be used to bypass some of the aforementioned limitations. For example, the femora6, feather buds7,8, and limbs9 of the chicken have all been studied using tissue culturing methods, as have the testes of the mouse10 and the roots and stems of plants11. These methods grant scientists a high degree of control over the tissue development, such as the ability to fluctuate the temperature and alter the nutrient availability. The isolation of the tissue from the whole embryo also makes it far less susceptible to the lethal effects of chemicals, thus enabling manipulation studies on a global scale at higher concentrations. Another notable advantage of in vitro culturing is the preservation of the tissue&#39;s cellular environment; the arrangement of tissues remains relatively unchanged, making it possible to study the interactions between different tissue types9. Thus, in vitro culturing opens doors to additional experimental approaches not available in in vivo or in ovo models.
Currently, studying the development of the embryonic chicken eye using chemicals is particularly challenging. A number of extraembryonic membranes cover the embryo, making it difficult to apply microbeads or chemicals; the embryo is also very active within the egg as it gets older, further complicating an already difficult method. This protocol enables easy access to the eye and its surrounding tissues, eliminating these barriers, while also providing new opportunities to examine the developmental processes within the eye. This protocol was established to study the induction of the scleral ossicles within the embryonic eye.

<table><tbody><tr><td>35 mm cell culture petri dishes</td><td>Corning</td><td>353001</td><td>easy grip tissue culture dish, polystyrene, non-pyrogenic</td></tr><tr><td>100 mm cell culture petri dishes</td><td>Corning</td><td>353003</td><td>tissue culture dish, polystyrene, non-pyrogenic</td></tr><tr><td>paper tissue</td><td>Kimtech</td><td>34155</td><td>Kimtech Science Brand Task Wipers, 280 per carton</td></tr><tr><td>wire mesh</td><td>n/a</td><td>n/a</td><td>stainless steel wire mesh (grid size 0.7 mm)</td></tr><tr><td>disposable glass pipettes</td><td>VWR</td><td>14673-010</td><td>Borosilicate glass disposable 5 3/4&quot;</td></tr><tr><td>nutrient medium</td><td>Gibco</td><td>12591-038</td><td>Fitton-Jackson Modification, [+] L-glutamine with phenol red (BGJB)</td></tr><tr><td>penicillin-streptomycin</td><td>Sigma-Aldrich</td><td>P4458</td><td>10000 units/mL penicillin streptomycin solution stabilised</td></tr><tr><td>filter paper</td><td>Whatman</td><td>1454 090</td><td>semi-porous filter paper 90mm</td></tr><tr><td>fertilized chicken eggs</td><td>Dalhousie University Agricultural College</td><td>n/a</td><td>can be obtained from local farms</td></tr><tr><td>sodium chloride (NaCl)</td><td>EMD</td><td>SX0420-3</td><td>sodium chloride crystals, reagent grade</td></tr><tr><td>1 L glass bottle</td><td>VWR</td><td>89000-240</td><td>1 L pyrex autoclavable glass bottle</td></tr><tr><td>ethanol</td><td>Fisher Scientific</td><td>BP82011</td><td>70% molecular biology grade</td></tr><tr><td>tupperware containers</td><td>n/a</td><td>n/a</td><td>store-bought and sterilized with EtOH</td></tr><tr><td>disposable razor blades</td><td>VWR</td><td>55411-050</td><td>single edge industrial razor blades (surgical carbon steel)</td></tr><tr><td>plastic spoons</td><td>n/a</td><td>n/a</td><td>store-bought and sterilized with EtOH</td></tr><tr><td>dust mask</td><td>3M</td><td>n/a</td><td>3M 8500 Comfort Mask</td></tr><tr><td>paraformaldehyde</td><td>Sigma-Aldrich</td><td>P6148</td><td>paraformaldehyde, reagent grade, crystalline</td></tr><tr><td>neutral-buffered formalin</td><td>Fisher Scientific</td><td>72210</td><td>10% neutral buffered formalin</td></tr><tr><td>phosphate buffered saline (PBS)</td><td>n/a</td><td>n/a</td><td>10X phosphate buffered saline pH 7.4 (137mM NaCl, 2.5mM KCl, 4.3mM Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;, 1.4mM KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;)</td></tr><tr><td>15 ml falcon tubes</td><td>VWR</td><td>21008-216</td><td>presterilized centrifuge tubes</td></tr><tr><td>forceps</td><td>FST</td><td>n/a</td><td>fine forceps</td></tr><tr><td>chick saline&amp;nbsp;</td><td>n/a</td><td>n/a&amp;nbsp;</td><td>0.85% NaCl</td></tr><tr><td>tinfoil</td><td>n/a</td><td>n/a</td><td>store-bought</td></tr><tr><td>paper towel</td><td>n/a</td><td>n/a</td><td>store-bought</td></tr></tbody></table>

organotypic culture method to study the development of embryonic chicken tissues

The embryonic chicken is commonly used as a reliable model organism for vertebrate development. Its accessibility and short incubation period makes it ideal for experimentation. Currently, the study of these developmental pathways in the chicken embryo is conducted by applying inhibitors and drugs at localized sites and at low concentrations using a variety of methods. In vitro tissue&#160;culturing is a technique that enables the study of tissues separated from the host organism, while simultaneously bypassing many of the physical limitations present when working with whole embryos, such as the susceptibility of embryos to high doses of potentially lethal chemicals. Here, we present an organotypic culturing protocol for culturing the embryonic chicken half head in vitro, which presents new opportunities for the examination of developmental processes beyond the currently established methods.

Using this method, an embryonic chicken eye can be cultured from day 8 of its development (HH34) in vitro for 4 days. Four days of in ovo development corresponds to HH38.
This culturing method supports the development of feather buds surrounding the eye and&#160;on the eyelids (Figure 1B). These feather buds are not present in ovo at HH34 prior to the culturing (<strong cl...

Watch this Scientific Journal Video about Organotypic Culture Method to Study the Development Of Embryonic Chicken Tissues at JoVE.com

Organotypic Culture Method to Study the Development Of Embryonic Chicken Tissues

Here, we present an organotypic culturing protocol to grow embryonic chicken organs in vitro. Using this method, the development of embryonic chicken tissue can be studied, while maintaining a high degree of control over the culture environment.

The embryonic chicken is commonly used as a reliable model organism for vertebrate development. Its accessibility and short incubation period makes it ...

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Developmental Biology

5.9K Views.  Mount Saint Vincent University. Here, we present an organotypic culturing protocol to grow embryonic chicken organs in vitro. Using this method, the development of embryonic chicken tissue can be studied, while maintaining a high degree of control over the culture environment.

Video: Organotypic Culture Method to Study the Development Of Embryonic Chicken Tissues

Optimized Ex-ovo Culturing of Chick Embryos to Advanced Stages of Development

Research in anatomy, embryology, and developmental biology has largely relied on the use of model organisms. In order to study development in live embryos model organisms, such as the chicken, are often used. The chicken is an excellent model organism due to its low cost and minimal maintenance, however they present observational challenges because they are enclosed in an opaque eggshell. In order to properly view the embryo as it develops, the shell must be windowed or removed. Both windowing and ex ovo techniques have been developed to assist researchers in the study of embryonic development. However, each of the methods has limitations and challenges. Here, we present a simple, optimized ex ovo culture technique for chicken embryos that enables the observation of embryonic development from stage HH 19 into late stages of development (HH 40), when many organs have developed. This technique is easy to adopt in both undergraduate classes and more advanced research laboratories where embryo manipulations are conducted.

Optimized Ex-ovo Culturing of Chick Embryos to Advanced Stages of Development

Viewing and accessing the chicken embryo during development can be challenging. We have developed an ex ovo method that is simple, cost effective, and can easily be used in a classroom or research setting. This method provides access to the embryo into late stages of embryonic development (HH 40).

Research in anatomy, embryology, and developmental biology has largely relied on the use of model organisms. In order to study development in live embryos ...

A Submerged Filter Paper Sandwich for Long-term Ex Ovo Time-lapse Imaging of Early Chick Embryos

Due to its availability, low cost, flat geometry, and transparency, the ex ovo chick embryo has become a major vertebrate animal model for the study of morphogenetic events, such as gastrulation2, neurulation3-5, somitogenesis6, heart bending7,8, and brain formation9-13, during early embryogenesis. Key to understanding morphogenetic processes is to follow them dynamically by time-lapse imaging. The acquisition of time-lapse movies of chick embryogenesis ex ovo has been limited either to short time windows or to the need for an incubator to control temperature and humidity around the embryo14. Here, we present a new technique to culture chick embryos ex ovo for high-resolution time-lapse imaging using transmitted light microscopy. The submerged filter paper sandwich is a variant of the well-established filter paper carrier technique (EC-culture)1 and allows for the culturing of chick embryos without the need for a climate chamber. The embryo is sandwiched between two identical filter paper carriers and is kept fully submerged in a simple, temperature-controlled medium covered by a layer of light mineral oil. Starting from the primitive streak stage (Hamburger-Hamilton stage 5, HH5)15 up to at least the 28-somite stage (HH16)15, embryos can be cultured with either their ventral or dorsal side up. This allows the acquisition of time-lapse movies covering about 30 hr of embryonic development. Representative time-lapse frames and movies are shown. Embryos are compared morphologically to an embryo cultured in the standard EC-culture.
The submerged filter paper sandwich provides a stable environment to study early dorsal and ventral morphogenetic processes. It also allows for live fluorescence imaging and micromanipulations, such as microsurgery, bead implantation, microinjection, gene silencing, and electroporation, and has a strong potential to be combined with immersion objectives for laser-based imaging (including light-sheet microscopy).

A Submerged Filter Paper Sandwich for Long-term Ex Ovo Time-lapse Imaging of Early Chick Embryos

Here, we present a new technique to culture chick embryos ex ovo for time-lapse imaging using transmitted light and fluorescence microscopy. The submerged filter paper sandwich is a variant of the well-established filter paper carrier technique1 that allows for the culturing of chick embryos fully-submerged in a liquid culture medium.

Due to its availability, low cost, flat geometry, and transparency, the ex ovo chick embryo has become a major vertebrate animal model for the study of ...

Method for Culture of Early Chick Embryos ex vivo (New Culture)

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  the formation and patterning of brain, neural tube, somite and heart primordia. Applications of chick embryo culture include electroporation of DNA or RNA constructs in order to analyze gene function, grafts of growth factor coated beads such as FGFs and BMPs , as well as whole mount in situ hybridization and immunohistochemistry. This video demonstrates the different steps in chick embryo culture; First, the embryo is explanted in saline. Then, the embryo is centered on a glass ring. The membranes surrounding the embryo are lifted along the walls of the ring. The ring is then placed in a culture dish containing a pool of albumine. The culture dish is sealed and placed in a humid chamber, where the embryo is cultured for up to 24 hrs. Finally, the embryo is removed from the ring, fixed and processed for further applications. A troubleshooting guide is also presented.

Method for Culture of Early Chick Embryos ex vivo New Culture

This video demonstrates New culture, a method by which chick embryos are cultured outside the egg for up to 24 hr. This method enables one to study early development (primitive streak to 14 som.), a period corresponding to E7-9 in mouse. Applications of this technique include electroporation, in situ hybridization and immunohistochemistry.

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

Biology

Two-step Approach to Explore Early- and Late-stages of Organ Formation in the Avian Model: The Thymus and Parathyroid Glands Organogenesis Paradigm

The avian embryo, as an experimental model, has been of utmost importance for seminal discoveries in developmental biology. Among several approaches, the formation of quail-chicken chimeras and the use of the chorioallantoic membrane (CAM) to sustain the development of ectopic tissues date back to the last century. Nowadays, the combination of these classical techniques with recent in vitro methodologies offers novel prospects to further explore organ formation.
Here we describe a two-step approach to study early- and late-stages of organogenesis. Briefly, the embryonic region containing the presumptive territory of the organ is isolated from quail embryos and grown in vitro in an organotypic system (up to 48 h). Cultured tissues are subsequently grafted onto the CAM of a chicken embryo. After 10 days of in ovo development, fully formed organs are obtained from grafted tissues. This method also allows the modulation of signaling pathways by the regular administration of pharmacological agents and tissue genetic manipulation throughout in vitro and in ovo developmental steps. Additionally, developing tissues can be collected at any time-window to analyze their gene-expression profile (using quantitative PCR (qPCR), microarrays, etc.) and morphology (assessed with conventional histology and immunochemistry).
The described experimental procedure can be used as a tool to follow organ formation outside the avian embryo, from the early stages of organogenesis to fully formed and functional organs.

This article provides an updated approach to the classical quail-chicken chimera system to study organ formation, by combining novel in vitro and in ovo experimental procedures.

The avian embryo, as an experimental model, has been of utmost importance for seminal discoveries in developmental biology. Among several approaches, the ...

Chicken Embryo Spinal Cord Slice Culture Protocol

Slice cultures can facilitate the manipulation of embryo development both pharmacologically and through gene manipulations. In this reduced system, potential lethal side effects due to systemic drug applications can be overcome. However, culture conditions must ensure that normal development proceeds within the reduced environment of the slice. We have focused on the development of the spinal cord, particularly that of spinal motor neurons. We systematically varied culture conditions of chicken embryo slices from the point at which most spinal motor neurons had been born. We assayed the number and type of motor neurons that survived during the culture period and the position of those motor neurons compared to that in vivo. We found that serum type and neurotrophic factors were required during the culture period and were able to keep motor neurons alive for at least 24 hr and allow those motor neurons to migrate to appropriate positions in the spinal cord. We present these culture conditions and the methodology of preparing the embryo slice cultures using eviscerated chicken embryos embedded in agarose and sliced using a vibratome.

Slice cultures facilitate the manipulation of embryo development by gene and pharmacological perturbations. However, culture conditions must ensure that normal development can proceed within the reduced environment of the slice. We illustrate a protocol that facilitates normal spinal cord development to proceed for at least 24 hr.

Slice cultures can facilitate the manipulation of  embryo development both pharmacologically and through gene manipulations. In  this reduced system, ...

Neuroscience

Isolation of Embryonic Tissues and Formation of Quail-Chicken Chimeric Organs Using The Thymus Example

The capacity to isolate embryonic tissues was an essential step for establishing the quail-chicken chimera system, which in turn has provided undisputed contributions to unveiling key processes in developmental biology.
Herein is described an optimized method to isolate embryonic tissues from quail and chickens by microsurgery and enzymatic digestion while preserving its biological properties. After isolation, tissues from both species are associated in an in vitro organotypic assay for 48 h. Quail and chicken tissues can be discriminated by distinct nuclear features and molecular markers allowing the study of the cellular cross-talk between heterospecific association of tissues. This approach is, therefore, a useful tool for studying complex tissue interactions in developmental processes with highly dynamic spatial modifications, such as those occurring during pharyngeal morphogenesis and the formation of the foregut endoderm-derived organs. This experimental approach was first developed to study the epithelial-mesenchymal interactions during early-stages of thymus formation. In this, the endoderm-derived prospective thymic rudiment and mesoderm-derived mesenchyme, were isolated from quail and chicken embryos, respectively.
The capacity of the associated tissues to generate organs can be further tested by grafting them onto the chorioallantoic membrane (CAM) of a chicken embryo. The CAM provides nutrients and allows gas exchanges to the explanted tissues. After 10 days of in ovo development, the chimeric organs can be analyzed in the harvested explants by conventional morphological methods. This procedure also allows studying tissue-specific contributions during organ formation, from its initial development (in vitro development) to the final stages of organogenesis (in ovo development).
Finally, the improved isolation method also provides three-dimensionally (3D) preserved embryonic tissues, that can also be used for high-resolution topographical analysis of tissue-specific gene-expression patterns.

This article provides a method to isolate pure embryonic tissues from quail and chicken embryos that can be combined to form ex vivo chimeric organs.

The capacity to isolate embryonic tissues was an essential step for establishing the quail-chicken chimera system, which in turn has provided undisputed ...

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

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.

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

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.

Understanding the relationships between genetic and microenvironmental factors that drive normal and malformed embryonic development is fundamental for ...

Efficient Vascularization of Kidney Organoids through Intracelomic Transplantation in Chicken Embryos

Kidney organoids derived from human induced pluripotent stem cells contain nephron-like structures that resemble those in the adult kidney to a certain degree. Unfortunately, their clinical applicability is hampered by the lack of a functional vasculature and consequently limited maturation in vitro. The transplantation of kidney organoids in the celomic cavity of chicken embryos induces vascularization by perfused blood vessels, including the formation of glomerular capillaries, and enhances their maturation. This technique is very efficient, allowing for the transplantation and analysis of large numbers of organoids. This paper describes a detailed protocol for the intracelomic transplantation of kidney organoids in chicken embryos, followed by the injection of fluorescently labeled lectin to stain the perfused vasculature, and the collection of transplanted organoids for imaging analysis. This method can be used to induce and study organoid vascularization and maturation to find clues for enhancing these processes in vitro and improve disease modeling.

Here, we present a detailed protocol for the transplantation of kidney organoids in the celomic cavity of chicken embryos. This method induces vascularization and enhanced maturation of the organoids within 8 days and can be used to study these processes in an efficient manner.

Kidney organoids derived from human induced pluripotent stem cells contain nephron-like structures that resemble those in the adult kidney to a certain ...

A Comprehensive Review of Avian Embryonic Skin Explant Culture Protocols

Using Avian Skin Explants to Study Tissue Patterning and Organogenesis

The developing avian skin during embryogenesis is a unique model that can provide valuable insights into tissue patterning. Here three variations on skin explant cultures to examine different aspects of skin development are described. First, ex vivo organ cultures and manipulations offer researchers opportunities to observe and study the development of feather buds directly. Skin explant culture can grow for 7 days enabling direct analysis of cellular behavior and 4D imaging at intervals during this growth period. This also allows for physical and molecular manipulations of culture conditions to visualize tissue response. For example, growth factor-coated beads can be applied locally to induce changes in feather patterning in a limited area. Alternatively, viral transduction can be delivered globally in the culture media to up or downregulate gene expression. Second, the skin recombination protocol allows researchers to investigate tissue interactions between the epidermis and mesenchyme that are derived from different skin regions, different life stages, or different species. This affords an opportunity to test the time window in which the epithelium is competent to respond to signals and its ability to form different skin appendages in response to signals from different mesenchymal sources. Third, skin reconstitution using dissociated dermal cells overlaid with intact epithelium resets skin development and enables the study of the initial processes of periodic patterning. This approach also enhances our ability to manipulate gene expression among the dissociated cells before creating the reconstituted skin explant. This paper provides the three culture protocols and exemplary experiments to demonstrate their utility.

Author Spotlight: Insight into the Current Experimental Avian Skin Explant Methodologies

Here we describe protocols for three types of avian embryonic skin explant cultures that can be used to examine tissue interactions, 4D imaging timelapse movie (3D plus time), global or local perturbation of molecular function, and systems biology characterization.

The developing avian skin during embryogenesis is a unique model that can provide valuable insights into tissue patterning. Here three variations on skin ...

Grafting Human Brain Organoids into the Chorioallantoic Membrane of the Chick Embryo

Early Unguided Human Brain Organoid Neurovascular Niche Modeling into the Permissive Chick Embryo Chorioallantoic Membrane

Engrafting organoids into vascularized tissues in model animals, such as the immunodeficient mouse or chick embryo chorioallantoic membrane (CAM), has proven efficient for neovascularization modeling. The CAM is a richly vascularized extraembryonic membrane, which shows limited immunoreactivity, thus becoming an excellent hosting model for human origin cell transplants.
This paper describes the strategy to engraft human brain organoids differentiated at multiple maturation stages into the CAM. The cellular composition of brain organoids changes with time, reflecting the milestones of human brain development. We grafted brain organoids at relevant maturation stages: neuroepithelial expansion (18 DIV), early neurogenesis (60 DIV), and early gliogenesis (180 DIV) into the CAM of embryonic day (E)7 chicken embryos. Engrafted brain organoids were harvested 5 days later and their histological features were analyzed.
No histological signs of neovascularization in the grafted organoids or abnormal blood vessels adjacent to the graftings were detected. Moreover, remarkable changes were observed in the cellular composition of the grafted organoids, namely, an increase in the number of glial fibrillary acidic protein-positive-reactive astrocytes. However, the cytoarchitectural changes were dependent on the organoid maturation stage. Altogether, these results suggest that brain organoids can grow in the CAM, and they show differences in the cytoarchitecture depending on their maturation stage at grafting.

Author Spotlight: Optimizing the Neurovascular Development of Human Brain Organoid in Chick Embryo

Here, we present a protocol to engraft human brain organoids at multiple maturation stages into the chick chorioallantoic membrane (CAM). Brain organoids were grown following unguided standardized protocols.

Engrafting organoids into vascularized tissues in model animals, such as the immunodeficient mouse or chick embryo chorioallantoic membrane (CAM), has ...

Organotypic Culture Method to Study the Development Of Embryonic Chicken Tissues

Summary

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Chapters in this video

Optimized Ex-ovo Culturing of Chick Embryos to Advanced Stages of Development

A Submerged Filter Paper Sandwich for Long-term Ex Ovo Time-lapse Imaging of Early Chick Embryos

Method for Culture of Early Chick Embryos ex vivo (New Culture)

Two-step Approach to Explore Early- and Late-stages of Organ Formation in the Avian Model: The Thymus and Parathyroid Glands Organogenesis Paradigm

Chicken Embryo Spinal Cord Slice Culture Protocol

Isolation of Embryonic Tissues and Formation of Quail-Chicken Chimeric Organs Using The Thymus Example

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

Efficient Vascularization of Kidney Organoids through Intracelomic Transplantation in Chicken Embryos

Using Avian Skin Explants to Study Tissue Patterning and Organogenesis

Early Unguided Human Brain Organoid Neurovascular Niche Modeling into the Permissive Chick Embryo Chorioallantoic Membrane