The authors would like to thank J. Kueper and J. Wittschier for excellent technical assistance and Prof. Ruebben for generously supplying tumor material.

All equipment and reagents have to be purchased sterile or needs to be heat or steam sterilized or sterilized with 70% ETOH.
The authors state that experiments on animals were performed in accordance with the European Communities Council Directive (86/609/EEC), following the Guidelines of the NIH regarding the care and use of animals for experimental procedures and the regulations set forth by the Institutional Animal Care and Use Committee (IACUC) at the University of Duisburg-Essen (Germany).
Part 1: Incubation of eggs
<ol>
 <li>Fertilized eggs can be stored at 13&deg;C for up to one week.</li>
 <li>Eggs are incubated for 72 hours, lying horizontally in an incubator with a moving tray, rotating the eggs 12 times a day continuously. Humidity is kept at 60 - 62%, incubation temperature at 37.5&deg;C. 
Note: Before starting incubation, dirt, feathers and excrement are carefully removed from the egg shells mechanically by dry wiping with common, gray zigzag hand paper towels, which have a rough rather than a soft surface structure. Wiping the eggs with ethanol or disinfectant, however, significantly reduced survival rate of the embryos, irrespective of the liquid used.</li>
</ol>
Part 2: Ex ovo culture
<ol>
 <li>After incubation for 72 hours, eggs are removed from the incubator. 
Note: The eggs are incubated for 72 hours because - according to our experiments - for unknown reasons but in agreement with Auerbach et al.1 earlier development stages had significantly lower survival rates. At stages later than 72 hours the yolk sack which is not yet covered by the CAM becomes thinner and tends to adhere to the egg shell leading (i) to small haemorrhages at the area of adherence and (ii) the rupture of the yolk-sack membrane. Therefore the presented method is not applicable to embryos older than 72 hours.</li>
 <li>The top side of the eggs, where the embryo resides, is marked by pencil since the embryo resists rotation to a certain extent.</li>
 <li>The egg is held horizontally with the pencil mark on top and cracked on the edge of an 80 mm triangular magnetic stir bar laying oriented perpendicular to the long axis of the egg. Note: For a better feeling, no gloves should be used, but hands should be disinfected with 70% EtOH. It is important that the egg shell as well as the egg membrane are opened up. Leaking of egg white is a safe sign that the egg membrane has been perforated.</li>
 <li>The egg is kept close to the bottom of the Petri dish to avoid further leakage of egg white. 
Note: During the initial opening procedure, it is important that the egg white on the bottom of the Petri dish is still connected to the rest inside the egg as this results in a vacuum inside the egg which holds the yolk with the embryo on top inside the egg. The vacuum can be regulated either by finger pressure which controls the inlet of air into the egg or by lifting the egg which increases the column of connected egg white i.e. the vacuum.</li>
 <li>By gently applying pressure to the meridian running through the crack and equator of the egg by thumb and middle fingers it is possible to regulate the vacuum. The content of the egg can then be transferred to the Petri dish undamaged, if the egg with the yolk is gently lifted and both halves of the shell are carefully separated. The embryo and the yolk vessels should lie on top of an undamaged yolk. 
Note: If the pencil mark is not kept on top, the embryo might be hurt when cracking the shell. If the eggs are not cracked carefully enough or the embryos are older than 72 hours (see above), the yolk sack sticks to the shell and often ruptures. The same happens when the humidity of the incubator is lower than 60% or eggs are not rotated during incubation.</li>
 <li>Ex ovo cultures are returned to an incubator and kept at 37.5&deg; 38.2&deg;C and 60% humidity. CO2 or O2 supply is not necessary if the incubator is not closed hermetically (ventilation grid partially open!) and special Petri dishes with spacers between lid and dish are used. 
 Note: Moving of the embryos should be kept to a minimum during the first days of incubation. Too much agitation results in high mortality rates. From incubation day 9 onwards, the embryos are less sensitive to agitation. 
 Dead embryos should be removed immediately to avoid infections. 
 If cell culture incubators are used, which are hermetically closed, O2 supply is mandatory for the embryos not to die. Humidity is essential for the CAM not to dry out and if incubation temperature is lower than 37&deg;C, the embryos will develop too slowly.</li>
</ol>
Part 3: Application of substances for angiogenesis assays
<ol>
 <li>Autoclaved folded filter papers (5.5 mm in diameter) are punched by a hole puncher and used as supporting material (carrier) for liquid substances applied to the CAM. 
 Note: Nearly each carrier material (see Table below) caused irritation of the CAM, especially when applied before incubation day 10, as it changed the developing CAM vessel architecture. This also included potentially inert substances like hydroxyethyl cellulose foil, Teflon sponges (PTFE-pledgets), collagen sponges, collagen foil, sterile gauze (swabs), round cover slips, and silicon rings, which all caused false positive results. Filter paper seemed to cause the least irritation and was therefore used in our assays.</li>
 <li>When the CAM is fully developed, up to 6 different samples may be applied and their effects can be compared on a single CAM.</li>
 <li>To avoid drying filters additional doses (3 &mu;g native angiotropin) or buffer (5 &mu;l PBS) should be re-applied every day.</li>
 <li>From day 3 after application, blood vessels orientate towards the angiogenic substances on the filters, whereas the pattern of blood vessels surrounding controls is unchanged. 
 Note: The CAM should not be mistaken for the yolk-sack. The CAM is a dynamic developing structure while the yolk sack is static with little variances during development. On developing day three the developing CAM can be recognized for the first time at the caudal end of the embryo as a small vesicle. Between day three and day ten the initial vesicle expands, creating a folded two-layered membrane overgrowing the yolk sack at an increasing rate. The application site should be half way between embryo and the fold of the CAM (border) as otherwise the embryo hinders microscopic observation of effects in transmitted light. Alternatively, the filter with the angiogenic substance is internalized when the CAM keeps growing.</li>
</ol>
Part 4: Inoculation of cells onto the CAM
4.1.Preparation of cells
<ol>
 <li>Confluent cells are transferred from culture flask to FALCON tubes by the use of a sterile 25 ml plastic pipette using a pipette aid.</li>
 <li>To estimate cell number, 15 &mu;l of the cell suspension is transferred to a Neubauer chamber and cells are counted manually under a microscope. </li>
 <li>Falcon tubes are centrifuged for 3 min at 1000 rpm at room temperature and the supernatant is discarded.</li>
 <li>For live cell tracking, cells are diluted with PBS to the desired concentration (here: 2x107 cells/ml) and stained with e.g. PKH26 (Sigma), a red fluorescent live cell membrane labelling kit, according to the manufacturers' protocol. 
Cells are re-suspended in cell culture medium (preferentially Dulbecco's modified eagle's medium; DMEM) to the desired concentration for injection (107 cell/ml) or inoculation (3x106 cell/ml). Different cell concentrations are applied for injections vs. inoculation according to concentrations already published in the literature15-17 and our experience.</li>
</ol>
4.2 Inoculation of cells onto the CAM
<ol>
<li>Rings (~ 1mm thickness) are cut from a 1ml pipette tip or a peripheral venous catheter (PVL) tube by the use of a sharp scalpel and applied to the CAM of an E6-E14 chick embryo cultured ex ovo. 
 Note: Try not to "saw" several times but to cut through the pipette tip or catheter tube with one cut using a new, very sharp scalpel to avoid sharp edges on the resulting rings. Check the rings under the microscope for sharp edges resulting from micro-cracks.</li>
<li>Depending on the size of the rings 20 &mu;l of 3x106 cell/ml cells are pipetted in total into one ring or as split volumes into multiple rings. 
 Note: In some cases traumatisation of the CAM by gentle scraping is a pre-requisite, e.g. to facilitate inoculation of cells17 and capillarisation of grafts (see below). If, however, traumatisation of the CAM is not intended or contraindicative, e.g. for studies of the invasive potential of tumor cells, rings should not be used for inoculation, but cells should be applied directly as application of rings or rings themselves might induce micro lesion. If rings are used, these should be cut as thin as possible to minimize weight and avoid indentation of the CAM.</li>
</ol>
Part 5: Intravitreous injection of cells
<ol>
 <li>A Hamilton microliter syringe is rinsed several times with 70% EtOH and finally with sterile PBS.</li>
 <li>The micro syringe is filled with the prepared stained or unstained cell suspension (see Part 4.1). 
Note: The injection site has to be carefully chosen, not to injure blood vessels of the CAM or adjacent structures of the embryo (brain!) by over penetration with the needle. The injection should be done under a dissection binocular. </li>
 <li>Under microscope control, carefully, but quickly penetrate the CAM and eye layers with the syringe needle and continuously inject 10 to maximal 20 &mu;l cell suspension (preferentially diluted in PBS) into the vitreous of the chick embryo cultured ex ovo. 
Note: If the CAM can not be penetrated with the needle, it might be removed at a site with few or no blood vessels by gently tearing it apart with two forceps.</li>
 <li>The needle should remain 1-2 seconds into the eye to avoid a loss of the injected cell suspension by leakage.</li>
</ol>
Part 6: Dissection of chicken limb buds
<ol>
 <li>Eggs are removed from the incubator after 3 to 4 days of incubation (E3/E4 = Hamburger Hamilton (HH) stage18-23) </li>
 <li>Eggs are cracked on the edge of a triangular magnetic stir bar and transferred into a Petri dish (details: see Part 2) and the embryo is dissected from the connected yolk vessels by cutting a circle using spring scissors.</li>
 <li>The embryo is transferred to a fresh Petri dish filled with cold, sterile PBS by use of a small drain spoon and washed by kind agitation.</li>
 <li>The embryo is transferred to a small Petri dish filled with sterile PBS and freed from surrounding membranes, carefully tearing them apart with fine forceps.</li>
 <li>Limb buds are detached by microsurgical forceps as close to the body as possible, grasped and transferred to the CAM of an 8-10-day-old host chicken cultured ex ovo (see grafting procedure).</li>
</ol>
Part 7: Preparation of mouse limb buds
<ol>
 <li>Time pregnant matings are set up and the morning of the day on which a vaginal plug is detected in females mating is designated gestation day 0.</li>
 <li>The pregnant female is sacrificed by cervical dislocation when the development of the embryos reached embryonic day (E) 10 to 13 and fixed on a wax board.</li>
 <li>The abdominal wall is moistened with 70% ETOH, cut along the midline and the skin flaps are fixed laterally by pins.</li>
 <li>Both horns of the uterus are removed from the abdomen, detached and transferred to a beaker with cold PBS.</li>
 <li>The embryos are separated, transferred to a Petri dish and uterus wall and embryonic membranes are removed carefully by the use of forceps.</li>
 <li>Limb buds are cut off by use of spring scissors or detached by fine forceps as close to the body as possible, grasped and transferred to the CAM of 8-10-day-old host chicken cultured ex ovo (see grafting procedure).</li>
</ol>
Part 8: Collection of tumor samples
<ol>
 <li>Tumor biopsies are collected in 2 ml Eppendorf tubes filled with tumor cell-specific culture medium (here: Leibovitz's medium) and kept at room temperature.</li>
 <li>Tumor biopsies are cut into 1-2 mm3 pieces by sterile scalpels. 
Note: If tumor samples were too small, they might not attach to the CAM, if they were too big, the CAM might be injured and the embryo might die.</li>
 <li>A piece from the core of the biopsy sample is grafted onto the CAM of 8-10-day-old host chicken cultured ex ovo (see grafting procedure). 
 Note: Taking material from the surface of the biopsy increases the risk of contamination with muscle or connective tissue.</li>
</ol>
Part 9: Grafting procedure
<ol>
 <li>For grafting of limb buds or tumor specimens, the fully developed CAM of 8-10-day-old chick embryos cultured ex ovo is used. </li>
 <li>The grafts are ideally placed on the CAM near a Y bifurcation of a blood vessel.</li>
 <li>At the desired application site, the CAM is selectively traumatized by gentle scraping off (forceps) the upper peridermal part of the double epithelial layer, leaving the basal layer intact. 
 Note: It is essential to avoid bleeding or visible rupture of capillaries.</li>
 <li>The graft is transferred to the CAM with minimal PBS attached. Depending on the graft, it might be placed once or twice on a site of the CAM, where no final grafting is intended to remove excessive PBS and then finally grafted at the prepared traumatized CAM site.</li>
 <li>The graft is grasped by fine forceps and layered onto the CAM. Depending on the sensitivity of the graft, light pressure might be applied (not suitable for limb buds) to manoeuvre the graft into a resulting indentation of the CAM (suitable for tumor samples). 
 Note: As the chick immune system develops at embryonic day (E) 18, ex ovo xenografts cultures should not be prolonged beyond E17 as the host chick embryos frequently die. 
 It is important to have available host CAMs, respectively chick embryos cultured ex ovo, timed to coincide with a source of donor tissue of your choice.</li>
</ol>
Part 10: Re-grafting
<ol>
 <li>The host chick embryo is killed by decapitation.</li>
 <li>The CAM with the attached graft is removed by a circular cut with pointed scissors and transferred to a Petri dish filled with PBS.</li>
 <li>Excessive CAM is cut from the graft by spring scissors except for the former attachment site.</li>
 <li>In case of bigger tumor samples, the graft is cut in halves or several smaller pieces and re-grafted with the cutting edge facing the new CAM of the second host.</li>
</ol>
Part 11: Representative Results
<img src="/files/ftp_upload/1620/1620fig1.jpg" alt="Figure 1" /> Figure 1. Shell-less chicken culture 
Chicken embryos of different development stages cultured ex ovo in Petri dishes.
<img src="/files/ftp_upload/1620/1620fig2.jpg" alt="Figure 2" /> Figure 2. Ex ovo CAM angiogenesis assay 
Fertilized chicken eggs were incubated horizontally at 37 &deg;C and humidity of 60 - 62% and cracked into Petri dishes at embryonic day 4 (E4). Incubation was continued under the same conditions. At E10 sterile filter papers (5.5 mm in diameter) were layered on top of the CAM and soaked either with 5 &mu;l PBS or 3 &mu;g native angiotropin6. Capillaries showed obvious alignment towards the angiotropin soaked filter paper at E14.
<img src="/files/ftp_upload/1620/1620fig3.jpg" alt="Figure 3" /> Figure 3. Ex ovo grafting of limb buds 
Limb buds from chick (E3/E4) and mouse (E13) were grafted onto the CAM of shell-less chicken cultures. Blood vessels from the CAM entered the limb buds after two days. The development of the limb buds continued in these ex ovo cultures reaching a two phalange stage in chick and displaying reduction of the interdigital webs in murine limb buds.
<img src="/files/ftp_upload/1620/1620fig4.jpg" alt="Figure 4" /> Figure 4. Ex ovo inoculation of tumor sample 
A biopsy sample of bladder carcinoma was inoculated onto the CAM of a 10-day-old chick embryo cultured ex ovo. After two days in culture, the tumor specimen was connected to the CAM vasculature and after 7 days, multiple blood vessels entering the graft were observed.

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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chick+embryo+chorioallantoic+membrane+models+to+quantify+angiogenesis+induced+by+inflammatory+and+tumor+cells+or+purified+effector+molecules.">Chick embryo chorioallantoic membrane models to quantify angiogenesis induced by inflammatory and tumor cells or purified effector molecules.</a> Methods Enzymology. 444, 21-44 (2008).</li><li>Ribatti, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chick+embryo+chorioallantoic+membrane+as+a+useful+tool+to+study+angiogenesis.">Chick embryo chorioallantoic membrane as a useful tool to study angiogenesis.</a> Int. Rev. Cell Mol. Biol. 270, 181-224 (2008).</li><li>H&#246;ckel, M., Sasse, J., Wissler, J. 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Res. 20, 237-239 (1992).</li><li>Demir, R., Naschberger, L., Demir, I., Melling, N., Dimmler, A., Papadopoulus, T., Sturzl, M., Klein, P., Hohenberger, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia+generates+a+more+invasive+phenotype+of+tumour+cells:+an+in+vivo+experimental+setup+based+on+the+chorioallantoic+membrane.">Hypoxia generates a more invasive phenotype of tumour cells: an in vivo experimental setup based on the chorioallantoic membrane.</a> Pathol. Oncol. Res. , (2008).</li><li>Kunzi-Rapp, K., Genze, F., Kufer, R., Reich, E., Hautmann, R. E., Gschwend, J. 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Innovative or just another chick culture protocol?
With former shell-less culture protocols1-4 the total survival rates of the chick embryos were low, e.g. ca. 30%1. The refined ex ovo culture protocol described in this video article, by contrast, allows for survival rates over 50%. Compared to classical in ovo cultures ex ovo culture of chick embryos significantly facilitates the accessibility of the CAM and chick embryo and enables their expe...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Fertilized Eggs</td>
<td>Animal</td>
<td>S&ouml;ries Trockels, Germany</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Mice</td>
<td>Animal</td>
<td>Charles Rivers Laboratories</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Incubator Type 3000 with rotating egg tray</td>
<td>Tools</td>
<td>Siepmann GmbH Germany</td>
<td>9503</td>
<td>Maintain at 37&deg;C with relative humidity set above 60%</td>
</tr>
<tr>
<td>Egg incubator BSS 160</td>
<td>Tools</td>
<td>Grumbach, Germany</td>
<td>8101</td>
<td>Maintain at 37-387deg;C with relative humidity set above 60%</td>
</tr>
<tr>
<td>PBS</td>
<td>Reagent</td>
<td>Sigma</td>
<td>&nbsp;</td>
<td>PBS should be cold (&gt; 4&deg;C) and sterile
</td>
</tr>
<tr>
<td>Dulbecco's modified eagle's medium</td>
<td>Reagent</td>
<td>Sigma</td>
<td>D 6429</td>
<td>DMEM culture medium</td>
</tr>
<tr>
<td>Leibovitz's L-15 Medium</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>31415-029</td>
<td>here: collection medium for tumor samples</td>
</tr>
<tr>
<td>Tumor cell -specific medium</td>
<td>Reagent</td>
<td>Sigma</td>
<td>&nbsp;</td>
<td>each research group should use the medium they culture their tumors cells in or common culture media, e.g. Leibovitz's Medium or Dulbecco's modified eagle's medium supplemented with 15% fetal bovine serum (FBS), 4mM L-glutamine and 50 &mu;M insulin</td>
</tr>
<tr>
<td>70% EtOH</td>
<td>Reagent</td>
<td>Sigma</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Magnetic stir bar, triangled 80 mm</td>
<td>Tool</td>
<td>VWR</td>
<td>442-0391</td>
<td>A triangled magnetic stir bar serves well to crack the eggs shell.</td>
</tr>
<tr>
<td>Forceps DUMONT #5 </td>
<td>Tool</td>
<td>Fine Science Tools</td>
<td>11252-30</td>
<td>bevelled very fine shanks (0.05 mm x 0.02 mm tip)</td>
</tr>
<tr>
<td>Forceps DUMONT #7</td>
<td>Tool</td>
<td>Fine Science Tools</td>
<td>11271-30</td>
<td>curved shanks (0.07 mm x 0.10 mm tip)</td>
</tr>
<tr>
<td>Spring scissors, straight, 8cm</td>
<td>Tool</td>
<td>Fine Science Tools</td>
<td>15000-00</td>
<td>fine, small straight blades</td>
</tr>
<tr>
<td>Fine Iris scissors, straight</td>
<td>Tool</td>
<td>Fine Science Tools</td>
<td>14094-11</td>
<td>Use to cut our the CAM</td>
</tr>
<tr>
<td>Standard scissors, straight, sharp/blunt</td>
<td>Tool</td>
<td>Fine Science Tools</td>
<td>14007-14</td>
<td>Use for decapitation or cervical dislocation</td>
</tr>
<tr>
<td>microliter syringe 1702 TLLX, 25 &mu;l</td>
<td>Tool</td>
<td>Hamilton</td>
<td>80222</td>
<td>Use for injection into the vitreous</td>
</tr>
<tr>
<td>Hamilton 33-G needle (15 mm)</td>
<td>Tool</td>
<td>Fine Science Tools</td>
<td>18073-15</td>
<td>Use for injection into the vitreous</td>
</tr>
<tr>
<td>Sterile scalpels</td>
<td>Tool</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Use for dissecting tumor samples</td>
</tr>
<tr>
<td>Small drain spoon</td>
<td>Tool</td>
<td>Geuder, Germany</td>
<td>15758</td>
<td>Use to transfer of small chick embryos</td>
</tr>
<tr>
<td>Wax board with fixing pins</td>
<td>Tool</td>
<td>Self made</td>
<td>&nbsp;</td>
<td>Use to fix of animals for dissection</td>
</tr>
<tr>
<td>Petri dishes 60 x 15 mm</td>
<td>Tool</td>
<td>Greiner</td>
<td>628102</td>
<td>&nbsp;</td>
<tr>
<td>Petri dishes 92 x 16 mm</td>
<td>Tool</td>
<td>Sarstedt</td>
<td>82.1472</td>
<td>&nbsp;</td>
<tr>
<td>Sterile Petri dish 100 x 20 mm</td>
<td>Tool</td>
<td>Greiner</td>
<td>664160</td>
<td>extra deep, with spacers for ventilation</td>
</tr>
<tr>
<td>Beaker</td>
<td>Tool</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>300-600 ml</td>
</tr>
<tr>
<td>FALCON tubes</td>
<td>Tool</td>
<td>FALCON</td>
<td>&nbsp;</td>
<td>15 ml and 50 ml</td>
</tr>
<tr>
<td>Eppendorf tubes</td>
<td>Tool</td>
<td>Eppendorf</td>
<td>&nbsp;</td>
<td>1.5 ml and 2 ml</td>
</tr>
<tr>
<td>1ml pipette tips</td>
<td>Tool</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Use to cut plastic rings for application of substances / cells</td>
</tr>
<tr>
<td>Peripheral venous catheter (PVL)</td>
<td>Tool</td>
<td>Viggo&reg;</td>
<td>&nbsp;</td>
<td>Use to cut silicon rings from the tube for application of substances / cells</td>
</tr>
<tr>
<td>Pipettes and tips</td>
<td>Tool</td>
<td>Eppendorf / Abimed</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Autoclaved folded Filters 595 1/2, 110 mm diameter</td>
<td>Tool</td>
<td>Schleicher &amp; Schuell</td>
<td>311643</td>
<td>Carrier, which caused least irritation of the CAM; used in the paper</td>
</tr>
<tr>
<td>PTFE-Pledget, non resorbing, 6,3 x 152.4 x 1.6 mm</td>
<td>Tool</td>
<td>santec-medical</td>
<td>REF 277, LOT 832/511-1</td>
<td>Carrier; caused false positive results</td>
</tr>
<tr>
<td>Hxdroxyethylcellulose Tylose H 100000</td>
<td>Reagent</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Carrier mixed with Kollidon 17 PF; caused false positive results</td>
</tr>
<tr>
<td>Kollidon 17 PF</td>
<td>Reagent</td>
<td>BASF</td>
<td>S30200</td>
<td>mixed with Hydroxyethylcellulose; caused false positive results</td>
</tr>
<tr>
<td>Collagen Biomatrix TissuDura</td>
<td>Tool</td>
<td>Baxter Healthcare S.A.</td>
<td>REF B2246000999999</td>
<td>Carrier; caused false positive results</td>
</tr>
<tr>
<td>Round glass cover slips, 11 mm</td>
<td>Tool</td>
<td>Assistent Germany</td>
<td>1001/0011</td>
<td>Carrier; causes false positive results</td>
</tr>
<tr>
<td>Bovine Collagen Sponge</td>
<td>Tool</td>
<td>Wyeth</td>
<td>&nbsp;</td>
<td>Carrier; caused false positive results</td>
</tr>
<tr>
<td>Resodont Absorbable Collagen Membrane</td>
<td>Tool</td>
<td>Resorba Woundcare Germany</td>
<td>LOT 280303</td>
<td>Carrier; caused false positive results</td>
</tr>
<tr>
<td>Non-Woven Swabs</td>
<td>Tool</td>
<td>Fink &amp; Walter GmbH</td>
<td>REF 328132</td>
<td>Carrier; caused false positive results</td>
</tr>		</tbody>
		</table>
		</div>

chick ex ovo culture and ex ovo cam assay: how it really works

Chicken eggs in the early phase of breeding are between in vitro and in vivo systems and provide a vascular test environment not only to study angiogenesis but also to study tumorigenesis. After the chick chorioallantoic membrane (CAM) has developed, its blood vessel network can be easily accessed, manipulated and observed and therefore provides an optimal setting for angiogenesis assays. Since the lymphoid system is not fully developed until late stages of incubation, the chick embryo serves as a naturally immunodeficient host capable of sustaining grafted tissues and cells without species-specific restrictions. In addition to nurturing developing allo- and xenografts, the CAM blood vessel network provides a uniquely supportive environment for tumor cell intravasation, dissemination, and vascular arrest and a repository where arrested cells extravasate to form micro metastatic foci.
For experimental purposes, in most of the recent studies the CAM was exposed by cutting a window through the egg shell and experiments were carried out in ovo, resulting in significant limitations in the accessibility of the CAM and possibilities for observation and photo documentation of effects. When shell-less cultures of the chick embryo were used1-4, no experimental details were provided and, if published at all, the survival rates of these cultures were low. We refined the method of ex ovo culture of chick embryos significantly by introducing a rationally controlled extrusion of the egg content. These ex ovo cultures enhance the accessibility of the CAM and chick embryo, enabling easy in vivo documentation of effects and facilitating experimental manipulation of the embryo. This allows the successful application to a large number of scientific questions: (1) As an improved angiogenesis assay5,6, (2) an experimental set up for facilitated injections in the vitreous of the chick embryo eye7-9, (3) as a test environment for dissemination and intravasation of dispersed tumor cells from established cell lines inoculated on the CAM10-12, (4) as an improved sustaining system for successful transplantation and culture of limb buds of chicken and mice13 as well as (5) for grafting, propagation, and re-grafting of solid primary tumor tissue obtained from biopsies on the surface of the CAM14.
In this video article we describe the establishment of a refined chick ex ovo culture and CAM assay with survival rates over 50%. Besides we provide a step by step demonstration of the successful application of the ex ovo culture for a large number of scientific applications. 
Daniel S. Dohle, Susanne D. Pasa, and Sebastian Gustmann contributed equally to this study.

Watch this Scientific Journal Video about Chick ex ovo Culture and ex ovo CAM Assay: How it Really Works at JoVE.com

Chick ex ovo Culture and ex ovo CAM Assay: How it Really Works

The chick chorioallantoic membrane (CAM) is a unique, naturally immunodeficient supportive culture environment to study angiogenesis and tumorigenesis. This video article demonstrates the different steps in chick ex ovo culture, application of potentially angiogenic substances and successful inoculation of tumor cells and tissues on the surface of the CAM.

Chick ex ovo Culture and ex ovo CAM Assay: How it Really Works

Chicken eggs in the early phase of breeding are between in vitro and in vivo systems and provide a vascular test environment not only to study ...

chick-ex-ovo-culture-and-ex-ovo-cam-assay-how-it-really-works

Research

JoVE Journal

Biology

48.5K Views.  University of Duisburg-Essen. The chick chorioallantoic membrane (CAM) is a unique, naturally immunodeficient supportive culture environment to study angiogenesis and tumorigenesis. This video article demonstrates the different steps in chick ex ovo culture, application of potentially angiogenic substances and successful inoculation of tumor cells and tissues on the surface of the CAM.

Video: Chick ex ovo Culture and ex ovo CAM Assay: How it Really Works

In Ovo Electroporations of HH Stage 10 Chicken Embryos

Large size and external development of the chicken embryo have long made it a valuable tool in the study of developmental biology.  With the advent of molecular biological techniques, the chick has become a useful system in which to study gene regulation and function.  By electroporating DNA or RNA constructs into the developing chicken embryo, genes can be expressed or knocked down in order to analyze in vivo gene function.  Similarly, reporter constructs can be used for fate mapping or to examine putative gene regulatory elements.  Compared to similar experiments in mouse, chick electroporation has the advantages of being quick, easy and inexpensive.  This video demonstrates first how to make a window in the eggshell to manipulate the embryo.  Next, the embryo is visualized with a dilute solution of India ink injected below the embryo.  A glass needle and pipette are used to inject DNA and Fast Green dye into the developing neural tube, then platinum electrodes are placed parallel to the embryo and short electrical pulses are administered with a pulse generator.  Finally, the egg is sealed with tape and placed back into an incubator for further development.  Additionally, the video shows proper egg storage and handling and discusses possible causes of embryo loss following electroporation.

Chick in ovo electroporation is a technique which allows genetic manipulation of the avian embryo. Common applications of this technique include functional analysis of genes and putative enhancer elements. This video demonstrates neural tube electroporation in HH 10 chick embryos. Injection technique and proper egg handling are discussed.

Large size and external development of the chicken embryo have long made it a valuable tool in the study of developmental biology.  With the advent 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 ...

Deciphering Axonal Pathways of Genetically Defined Groups of Neurons in the Chick Neural Tube Utilizing in ovo Electroporation

Employment of enhancer elements to drive expression of reporter genes in neurons is a widely used paradigm for tracking axonal projection. For tracking axonal projection of spinal interneurons in vertebrates, germ line-targeted reporter genes yield bilaterally symmetric labeling. Therefore, it is hard to distinguish between the ipsi- and contra-laterally projecting axons. Unilateral electroporation into the chick neural tube provides a useful means to restrict expression of a reporter gene to one side of the central nervous system, and to follow axonal projection on both sides 1 ,2-5. This video demonstrates first how to handle the eggs prior to injection. At HH stage 18-20, DNA is injected into the sacral level of the neural tube, then tungsten electrodes are placed parallel to the embryo and short electrical pulses are administered with a pulse generator. The egg is sealed with tape and placed back into an incubator for further development. Three days later (E6) the spinal cord is removed as an open book preparation from embryo, fixed, and processed for whole mount antibody staining. The stained spinal cord is mounted on slide and visualized using confocal microscopy.

Deciphering Axonal Pathways of Genetically Defined Groups of Neurons in the Chick Neural Tube Utilizing in ovo Electroporation

This video demonstrates how to visualize axonal pathways of genetically defined groups of neurons in the embryonic chick spinal cord utilizing in ovo electroporation of reporter genes under the control of specific enhancer elements.

Employment of enhancer elements to drive expression of reporter genes in neurons is a widely used paradigm for tracking axonal projection. For tracking ...

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 ...

Ex Vivo Culture of Primary Human Fallopian Tube Epithelial Cells

Epithelial ovarian cancer is a leading cause of female cancer mortality in the United States. In contrast to other women-specific cancers, like breast and uterine carcinomas, where death rates have fallen in recent years, ovarian cancer cure rates have remained relatively unchanged over the past two decades 1. This is largely due to the lack of appropriate screening tools for detection of early stage disease where surgery and chemotherapy are most effective 2, 3. As a result, most patients present with advanced stage disease and diffuse abdominal involvement. This is further complicated by the fact that ovarian cancer is a heterogeneous disease with multiple histologic subtypes 4, 5. Serous ovarian carcinoma (SOC) is the most common and aggressive subtype and the form most often associated with mutations in the BRCA genes. Current experimental models in this field involve the use of cancer cell lines and mouse models to better understand the initiating genetic events and pathogenesis of disease 6, 7. Recently, the fallopian tube has emerged as a novel site for the origin of SOC, with the fallopian tube (FT) secretory epithelial cell (FTSEC) as the proposed cell of origin 8, 9. There are currently no cell lines or culture systems available to study the FT epithelium or the FTSEC. Here we describe a novel ex vivo culture system where primary human FT epithelial cells are cultured in a manner that preserves their architecture, polarity, immunophenotype, and response to physiologic and genotoxic stressors. This ex vivo model provides a useful tool for the study of SOC, allowing a better understanding of how tumors can arise from this tissue, and the mechanisms involved in tumor initiation and progression.

Ex Vivo Culture of Primary Human Fallopian Tube Epithelial Cells

The fallopian tube (FT) is emerging as an alternative site of origin for serous ovarian carcinoma (SOC). This protocol describes a novel method for the isolation and ex vivo culture of fallopian tube epithelial cells. This system recapitulates the in vivo epithelium and allows the study of SOC pathogenesis.

Epithelial ovarian cancer is a leading cause of female cancer mortality in the United States. In contrast to other women-specific cancers, like breast and ...

In Ovo Electroporation in Embryonic Chick Retina

Chicken embryonic retina is an excellent tool to study retinal development in higher vertebrates. Because of large size and external development, it is comparatively very easy to manipulate the chick embryonic retina using recombinant DNA/RNA technology. Electroporation of DNA/RNA constructs into the embryonic retina have a great advantage to study gene regulation in retinal stem/progenitor cells during retinal development. Different type of assays such as reporter gene assay, gene over-expression, gene knock down (shRNA) etc. can be performed using the electroporation technique. This video demonstrates targeted retinal injection and in ovo electroporation into the embryonic chick retina at the Hamburger and Hamilton stage 22-23, which is about embryonic day 4 (E4). Here we show a rapid and convenient in ovo electroporation technique whereby a plasmid DNA that expresses green fluorescent protein (GFP) as a marker is directly delivered into the chick embryonic subretinal space and followed by electric pulses to facilitate DNA uptake by retinal stem/progenitor cells. The new method of retinal injection and electroporation at E4 allows the visualization of all retinal cell types, including the late-born neurons1, which has been difficult with the conventional method of injection and electroporation at E1.52.

In Ovo Electroporation in Embryonic Chick Retina

The overall goal of this video is to show how to perform targeted retinal injection and in ovo electroporation of DNA/RNA constructs into the chick embryonic retina at the Hamburger and Hamilton stage 22-23, which is about embryonic day 4 (E4). This technique is very useful to study gene expression, gene regulation, and morphological change in developing chick retina.

Chicken embryonic retina is an excellent tool to study retinal development in higher vertebrates. Because of large size and external development, it is ...

Gene Transfer into Older Chicken Embryos by ex ovo Electroporation

The chicken embryo provides an excellent model system for studying gene function and regulation during embryonic development. In ovo electroporation is a powerful method to over-express exogenous genes or down-regulate endogenous genes in vivo in chicken embryos1. Different structures such as DNA plasmids encoding genes2-4, small interfering RNA (siRNA) plasmids5, small synthetic RNA oligos6, and morpholino antisense oligonucleotides7 can be easily transfected into chicken embryos by electroporation. However, the application of in ovo electroporation is limited to embryos at early incubation stages (younger than stage HH20 - according to Hamburg and Hamilton)8 and there are some disadvantages for its application in embryos at later stages (older than stage HH22 - approximately 3.5 days of development). For example, the vitelline membrane at later stages is usually stuck to the shall membrane and opening a window in the shell causes rupture of the vessels, resulting in death of the embryos; older embryos are covered by vitelline and allantoic vessels, where it is difficult to access and manipulate the embryos; older embryos move vigorously and is difficult to control the orientation through a relatively small window in the shell.
In this protocol we demonstrate an ex ovo electroporation method for gene transfer into chicken embryos at late stages (older than stage HH22). For ex ovo electroporation, embryos are cultured in Petri dishes9 and the vitelline and allantoic vessels are widely spread. Under these conditions, the older chicken embryos are easily accessed and manipulated. Therefore, this method overcomes the disadvantages of in ovo electroporation applied to the older chicken embryos. Using this method, plasmids can be easily transfected into different parts of the older chicken embryos10-12.

Gene Transfer into Older Chicken Embryos by ex ovo Electroporation

A method of gene transfer into chicken embryos at later incubation stages (older than Hamburger and Hamilton stage (HH) 22) is described. This method overcomes disadvantages of in ovo electroporation applied to older chicken embryos and is a useful technique to study gene function and regulation at older developmental stages.

The chicken embryo provides an  excellent model system for studying gene function and regulation during  embryonic development. In ovo electroporation is ...

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. Thus, protocols to isolate and culture individual organs of interest are essential to provide a method of both visualizing changes in development and allowing novel treatment strategies. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel. This substrate provides enough support to maintain the three-dimensional structure but is flexible enough to allow continued contraction. In brief, hearts are excised from the embryo and placed in a mixture of cold Matrigel diluted 1:1 with growth medium. After the diluted Matrigel solidifies, growth medium is added to the culture dish. Hearts excised as late as embryonic day 16.5 were viable for four days post-dissection. Analysis of the coronary plexus shows that this method does not disrupt coronary vascular development. Thus, we present a novel method for long-term culture of embryonic hearts.

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel.

Developmental studies in the mouse are hampered  by the inaccessibility of the embryo during gestation. Thus, protocols to  isolate and culture individual ...

Ex vivo Culture of Mouse Embryonic Skin and Live-imaging of Melanoblast Migration

Melanoblasts are the neural crest derived precursors of melanocytes; the cells responsible for producing the pigment in skin and hair. Melanoblasts migrate through the epidermis of the embryo where they subsequently colonize the developing hair follicles1,2. Neural crest cell migration is extensively studied in vitro but in vivo methods are still not well developed, especially in mammalian systems. One alternative is to use ex vivo organotypic culture3-6. Culture of mouse embryonic skin requires the maintenance of an air-liquid interface (ALI) across the surface of the tissue3,6. High resolution live-imaging of mouse embryonic skin has been hampered by the lack of a good method that not only maintains this ALI but also allows the culture to be inverted and therefore compatible with short working distance objective lenses and most confocal microscopes. This article describes recent improvements to a method that uses a gas permeable membrane to overcome these problems and allow high-resolution confocal imaging of embryonic skin in ex vivo culture6. By using a melanoblast specific Cre-recombinase expressing mouse line combined with the R26YFPR reporter line we are able to fluorescently label the melanoblast population within these skin cultures. The technique allows live-imaging of melanoblasts and observation of their behavior and interactions with the tissue in which they develop. Representative results are included to demonstrate the capability to live-image 6&#160;cultures in parallel.

Ex vivo Culture of Mouse Embryonic Skin and Live-imaging of Melanoblast Migration

We describe the dissection and ex vivo culture of mouse embryonic skin. The culture system maintains an air-liquid interface across the tissue surface and allows imaging on an inverted microscope. Melanoblasts, a component of the developing skin, are fluorescently labeled allowing their behavior to be observed using confocal microscopy.

Melanoblasts are the neural crest derived precursors of melanocytes; the cells responsible for producing the pigment in skin and hair. Melanoblasts ...

An Ex vivo Culture System to Study Thyroid Development

The thyroid is a bilobated endocrine gland localized at the base of the neck, producing the thyroid hormones T3, T4, and calcitonin. T3 and T4 are produced by differentiated thyrocytes, organized in closed spheres called follicles, while calcitonin is synthesized by C-cells, interspersed in between the follicles and a dense network of blood capillaries. Although adult thyroid architecture and functions have been extensively described and studied, the formation of the &#8220;angio-follicular&#8221; units, the distribution of C-cells in the parenchyma and the paracrine communications between epithelial and endothelial cells is far from being understood.
This method describes the sequential steps of mouse embryonic thyroid anlagen dissection and its culture on semiporous filters or on microscopy plastic slides. Within a period of four days, this culture system faithfully recapitulates in vivo thyroid development. Indeed, (i) bilobation of the organ occurs (for e12.5 explants), (ii) thyrocytes precursors organize into follicles and polarize, (iii) thyrocytes and C-cells differentiate, and (iv) endothelial cells present in the microdissected tissue proliferate, migrate into the thyroid lobes, and closely associate with the epithelial cells, as they do in vivo.
Thyroid tissues can be obtained from wild type, knockout or fluorescent transgenic embryos. Moreover, explants culture can be manipulated by addition of inhibitors, blocking antibodies, growth factors, or even cells or conditioned medium. Ex vivo development can be analyzed in real-time, or at any time of the culture by immunostaining and RT-qPCR.
In conclusion, thyroid explant culture combined with downstream whole-mount or on sections imaging and gene expression profiling provides a powerful system for manipulating and studying morphogenetic and differentiation events of thyroid organogenesis.

An Ex vivo Culture System to Study Thyroid Development

This protocol describes dissection of mouse embryonic thyroid anlagen and the culture of explants on semiporous filters or on microscopy plastic slides. This system is ideal to study morphogenetic or differentiation events occurring during thyroid development of wild type or knockout embryos, and is amenable to gain- and loss-of-function experiments.