The authors thank Mira Hryniuk, BA, for reviewing the English language of the article.

The use of human tissue was approved by the Institutional Review Board of the Catholic University of Louvain. The patients gave their written informed consent for the use of their ovarian tissue for research purposes.
1. Ordering day 0 eggs that are highly likely to be embryonated
<ol>
	<li>Find a certified laboratory-grade Lohman-selected white Leghorn egg supplier that reports high rates of embryonated eggs, which is primarily dependent on the age of the chicks.</li>
</ol>
2. Preparing the eggs for incubation
<ol>
	<li>Prior to the arrival of the eggs, assemble and equilibrate the egg incubator to 37 &#176;C in 40%-60% relative air humidity. Monitor the temperature and air humidity by inserting a thermometer and hygrometer into the incubator. Ensure the lid of the incubator is equipped with a glass window to allow for checking the internal parameters of the incubator without having to open it (Figure 1).</li>
	<li>Having received the day 0 eggs from Lohman-selected white Leghorn chicks from a certified laboratory-grade egg supplier, clean the surface of the shells with humidified paper, and dry them immediately. 
	NOTE: Since the eggshell membrane is porous, autoclaved water may be used to clean egg surfaces and control humidity within the incubator in order to minimize contamination risks.</li>
	<li>Label the eggs using a marker (e.g., date and egg number).</li>
	<li>Incubate the eggs with the pointed end facing down, and rotate them to allow the CAM to develop. Manually rotate the eggs by 180&#176; two to three times per day or use&#160;an automatic rotator. 
	NOTE: In order to ensure the eggs are indeed being rotated, the eggshell may be marked with an &#34;X&#34; and &#34;O&#34; on the two opposing lateral sides using a pencil or marker. The glass window in the lid of the incubator allows one to check the rotation of the eggs without opening the device.</li>
</ol>
3. Opening the eggshell on day 3 of ED
NOTE: A rectangular window is made in the eggshell on day 3 of ED.
<ol>
	<li>Prepare the laminar flow hood in order to work in sterile conditions. Place the following instruments under the hood, and disinfect them in 70% ethanol (if not already sterilized): 
	-Egg rack 
	-Egg candler or focal cold light source 
	-Marker 
	-Sterile straight pin 
	-Sterile 19 G needle 
	-5&#160;mL sterile syringe 
	-Scroll saw blade 
	-Sterile forceps 
	-Adhesive tape</li>
	<li>Transfer one egg from the incubator to the egg rack placed under the hood, and turn off the egg rotator.</li>
	<li>In the dark, identify the air pocket of the egg by placing the egg candler (or focal cold light source) against the eggshell. The air pocket is localized at the blunt end of the egg. Use a marker to pinpoint the center of the air pocket on the eggshell.</li>
	<li>Turn on the lights. Make a small hole in the eggshell where it is marked by gently rotating a sterile straight pin. A tiny opening of around 1 mm in diameter is usually sufficient. 
	NOTE: Be careful not to push the pin all the way through the egg. If this happens, discard the egg.</li>
	<li>Connect a sterile 19 G needle to a sterile 5 mL syringe.</li>
	<li>In the dark, locate the yolk sac using the egg candler (or focal cold light source), and puncture the egg through the hole made in step 3.4 with the sterile 19 G needle angled at 45&#176; toward the bottom of the egg; take great care not to disrupt the yolk sac. Aspirate between 1.5-2 mL of albumen to detach the CAM from the shell, and then close the hole with a piece of adhesive tape. 
	NOTE: If the yolk sac is disrupted during aspiration, the aspirated liquid in the syringe will be yellow-colored rather than transparent (albumen). If this happens, replace the needle and syringe. If a relatively large amount of egg yolk is sucked up, it may jeopardize the viability of the embryo.</li>
	<li>Turn on the lights. Place the egg horizontally, and draw a rectangular window measuring 1 cm x 1.5 cm with a marker. Do not make the window larger than the usual width of standard adhesive tape.</li>
	<li>Hold the egg in one hand, and gently saw the previously drawn&#160;window into the eggshell using a scroll saw blade. Ensure that the shell does not crack and do not&#160;cut all the way down to the depressed CAM. Blow regularly to remove shell dust and debris.</li>
	<li>Slide sterile forceps under the rectangular piece of sawed shell, and grasp deftly to remove it cleanly without damaging the CAM. Additionally, discard the white outer shell membrane to be able to see the embryo and its CAM. 
	NOTE: If some eggshell dust or debris falls onto the CAM, it may be removed using sterile forceps, being very careful not to tear the CAM.</li>
	<li>Identify viable embryonated eggs. They are discernible by their clear albumen and the vascular ring around the embryo, where a beating heart may sometimes be detected even at this stage (day 3 of ED). Discard non-fertilized or dead embryos.</li>
	<li>Place adhesive tape over the newly created window to avoid dehydration. Be sure to fold one end over itself for ease of removal.</li>
	<li>Put the egg back in the incubator with the opened window facing up and the tape not touching the CAM. Use a folded piece of paper or part of the egg rack to stop the egg from rolling. Make sure the rotating tray is off.</li>
	<li>Repeat steps 3.2-3.12 to open the remaining eggs.</li>
</ol>
4. Grafting frozen-thawed human ovarian tissue to the CAM
NOTE: Transplantation to the CAM should ideally be initiated between days 7-10 of ED.
<ol>
	<li>Thaw cryopreserved ovarian cortical strips under the laminar flow hood following the protocol described elsewhere30.</li>
	<li>Cut the cortical strips into three fragments of 4 mm x 2 mm x 1 mm. Use one piece as a non-grafted control and the other two for xenografting for 1 day or 6 days. 
	NOTE: If enough tissue is available, work in duplicate.</li>
	<li>Transfer one egg from the incubator to the egg rack positioned under the hood, with the window facing up.</li>
	<li>Peel the tape off the window, and ensure that the embryo is viable. At this stage, viable embryos feature extensive vasculature, a clear albumen, a visible heartbeat, and some embryo movement.</li>
	<li>Prepare the grafting site by gently traumatizing&#160;a small area of the CAM by laying a 1 cm2 strip of sterile ether-extracted lens paper onto the epithelial surface and removing it immediately. 
	NOTE: The CAM is an impenetrable barrier unless the membrane has been traumatized by the removal of the upper peridermal part of the double epithelial layer, leaving the basal layer intact. This technique also enhances the revascularization process by activating wound healing. If the sterile ether-extracted lens paper stays on the CAM too long, the membrane may adhere to the lens paper and tear. If this happens, discard the egg.&#160;</li>
	<li>Grasp one frozen-thawed ovarian cortical strip (4 mm x 2 mm x 1 mm) with microsurgical forceps, and place it onto the traumatized CAM with the medullary side against the CAM. Graft one tissue piece per egg.</li>
	<li>Cover the window with adhesive tape, and carefully return the egg to the incubator. Ensure that the eggshell opening sits upright and the egg is secure.</li>
	<li>Repeat steps 4.1-4.7 for the grafting of all the remaining tissues. 
	NOTE: The implants should be checked on each grafting day to assess the embryo viability and monitor changes in the vasculature.</li>
</ol>
5. Harvesting the grafts
NOTE: Xenografts should be harvested at the latest by day 17 of ED, since the immune system of the embryo becomes mature and competent from day 18.
<ol>
	<li>Place the egg on a rack, and enlarge the window in the eggshell to allow for better graft visualization and easier manipulation.</li>
	<li>Evaluate the graft macroscopically, and pay particular attention to the vascular reaction of the CAM towards the graft. Take digital photographs or videos for the record.</li>
	<li>Grasp the tissue or the surrounding CAM with forceps, and use scissors or a scalpel to excise the graft from the CAM carefully. 
	NOTE: The grafts become covered with a second layer of CAM at around day 3 of grafting and eventually become encapsulated. They may also have moved inside the egg by day 6, making it difficult to retrieve them in some cases.</li>
	<li>Analyze the excised tissues by any method appropriate for the given experiment.&#160;In the present study, tissue pieces were fixed in paraformaldehyde, paraffin-embedded and stained with hematoxylin and eosin following the steps outlined below:
	<ol>
		<li>Fix the fragments in 4% paraformaldehyde for 24 h, and embed them in paraffin using an automatic embedding device with the steps mentioned in Table 1.</li>
		<li>Leave the paraffin-embedded blocks overnight at 4&#176;C before cutting them into 5 &#181;m thick sections with a microtome.</li>
		<li>Spread the tissue sections on a glass slide placed on a hot plate (30 &#176;C), and leave them to dry for 2 h, followed by 24 h in an oven at 37 &#176;C.</li>
		<li>Afterwards, stain the slides with hematoxylin and eosin following a protocol described elsewhere31. Subsequently, digitize the samples using a slide scanner, and analyze them using an image analysis software.</li>
	</ol>
	</li>
</ol>

The authors have nothing to disclose.

The most challenging part of the protocol described here is making the small hole required to aspirate the albumen in order to detach the CAM from the eggshell prior to creating a window. Applying too much pressure can result in overpenetration or may even crack and destroy the egg, causing irrevocable damage to the CAM and its vasculature. To keep mistakes to a minimum during initial attempts to separate the CAM, it is strongly advised to practice making small holes in the eggshell of non-fertilized, grocery-bought eggs...

The demand for fertility preservation for oncological and benign indications, as well as social reasons, has dramatically increased over recent decades. However, various treatments used to cure malignant and non-malignant diseases are highly toxic to the gonads and can result in iatrogenic premature ovarian insufficiency, ultimately leading to infertility1. Established techniques for fertility preservation include embryo cryopreservation, immature or mature oocyte vitrification, and ovarian tissue cryopreservation2,3,4. Ovarian tissue freezing is the only available option for preserving fertility in prepubertal girls or women who require immediate cancer therapy. The restoration of endocrine function following ovarian tissue transplantation occurs in over 95% of subjects, with live birth rates ranging from 18% to 42%5,6,7,8,9.
Although the transplantation of frozen-thawed ovarian tissue has proven successful, there is still room for improvement. Indeed, as ovarian cortical fragments are transplanted without vascular anastomosis, they experience a period of hypoxia during which graft revascularization takes place10,11,12. The vast majority of studies investigating human ovarian tissue transplantation have used a xenografting model, in which ovarian tissue is transplanted to immunodeficient mice. The complete revascularization of the xenografts takes around 10 days, with both the host and graft vessels contributing to the formation of functional vessels12,13,14. Around 50%-90% of the follicle reserve is lost during this hypoxic window before the completion of graft revascularization10,15,16. It has been strongly suggested that this massive follicle loss occurs through both direct follicle death, as demonstrated by a decrease in the absolute follicle numbers left after grafting, and the activation of primordial follicle growth, as indicated by changes in follicle proportions towards increased rates of growing follicles17,18.
Interestingly, previous research works using various animal ovarian tissues grafted to chick chorioallantoic membrane (CAM), which has a constitution mimicking the typical grafting site of the peritoneum, have reported the inhibition of spontaneous follicle activation, with the primordial follicle reserve staying intact for up to 10 days19,20,21,22. Our team&#160;previously demonstrated that the grafting of frozen-thawed human ovarian tissue to CAM constituted a reliable approach for investigating human ovarian tissue transplantation in its first ischemic stages23&#160;and recently showed that this grafting method was able to counteract follicle activation24.
The CAM model is especially appealing not only because eggs are much cheaper than mice but also because of the highly vascularized nature of CAM, allowing scrutiny of the association between follicle activation and ovarian graft revascularization. The avian system is, indeed, one of the most common and versatile ways of studying angiogenesis25. Chick embryo development (ED) takes 21 days until hatching, and the CAM is formed within the first 4-5 days through the fusion of the allantois and chorion26. Notably, the chick embryo is a naturally immunodeficient host until day 17 of ED, so xenografting experiments can be performed without any risk of graft rejection27,28. Moreover, the CAM model approach does not raise any ethical or legal concerns in terms of European law29, making it an attractive alternative to other animal models. With regard to breeding conditions, chick embryos only need an incubator set at 37 &#176;C with a relative air humidity of 40%-60%. These limited experimentation requirements significantly reduce the research costs compared to use of immunodeficient mice.
The protocol presented herein aims to describe the development of a CAM xenografting model for human ovarian tissue and provide specific insights into the effectiveness of the technique, the time frame of graft revascularization, and the tissue viability over a 6 day grafting period. This protocol could be of great interest for investigating the mechanisms behind early post-grafting follicle loss and studying the impact of several agents (growth factors, hormones, etc.) on this phenomenon.

<table><tbody><tr><td>Agani hypodermic needle, 19 G</td><td>Terumo Europe</td><td>AN*1950R1</td><td>19 G needle to aspirate albumen</td></tr><tr><td>Terumo syringe, 5 mL concentric Luer lock</td><td>Terumo Europe</td><td>SS*05LE1</td><td>5-mL sterile syringe</td></tr><tr><td>Caseviewer v2.2</td><td>3DHISTECH</td><td></td><td>Image analysis software</td></tr><tr><td>Diethyl ether</td><td>Merck Chemicals</td><td>603-022-00-4</td><td>Sterile ether to traumatize the CAM</td></tr><tr><td>Eosin Y aqueous solution 0.5%</td><td>Merck</td><td>1098441000</td><td>Staining solution</td></tr><tr><td>Formaldehyde 4% aqueous solution buffered (Formalin 10%)</td><td>VWR</td><td>97139010</td><td>Formaldehyde used for tissue fixation</td></tr><tr><td>Fridge</td><td>Liebherr</td><td>7081260</td><td>Fridge at 4 &amp;deg;C used for paraffin-embedding</td></tr><tr><td>Heating plate</td><td>Schott</td><td>SLK2</td><td>Hot plate used to dry the slides</td></tr><tr><td>Incubator</td><td>Thermo Forma Scientific 3111</td><td>10365156</td><td>Oven used for slide incubation</td></tr><tr><td>Leica CLS 150 XE microscope cold light source</td><td>Leica</td><td>CLS 150 XE</td><td>Focal cold light source to candle the eggs</td></tr><tr><td>Lens cleaning tissue, grade 541</td><td>VWR</td><td>111-5003</td><td>Tissue to soak in sterile ether to traumatize the CAM</td></tr><tr><td>Mayer&#039;s hematoxylin</td><td>MERCK</td><td>1092491000</td><td>Staining solution</td></tr><tr><td>Methanol</td><td>VWR</td><td>20847307</td><td>Methanol</td></tr><tr><td>Microtome</td><td>ThermoScientific-MICROM</td><td>HM325-2</td><td>Microtome</td></tr><tr><td>Pannoramic P250 Flash III</td><td>3DHISTECH</td><td>/</td><td>Slide scanner at 20x magnification</td></tr><tr><td>Paraformaldehyde&amp;nbsp;</td><td>Merck</td><td>1,04,00,51,000</td><td>Paraffin-embedding solution</td></tr><tr><td>Paraplast Plus R</td><td>Sigma</td><td>P3683-1KG</td><td>Paraffin</td></tr><tr><td>Petri dish, 60x15 mm, sterile</td><td>Greiner</td><td>628161</td><td>Sterile petri dish</td></tr><tr><td>Pin holder</td><td>Fine Science Tools</td><td>26016-12</td><td>Pin holder</td></tr><tr><td>Polyhatch</td><td>Brinsea</td><td>CP01F</td><td>Egg incubator with automatic rotator</td></tr><tr><td>Scroll saw blade, 132 mm</td><td>Sencys</td><td>/</td><td>Saw blade to create a window in the eggshell</td></tr><tr><td>Stainless steel insert pins</td><td>Fine Science Tools</td><td>26007-02</td><td>Straight pin to make a hole in the eggshell</td></tr><tr><td>Steril-Helios&amp;nbsp;</td><td>Angelantoni Industrie</td><td>ST-00275400000</td><td>Laminar flow hood</td></tr><tr><td>Superfrost Plus bords rod&amp;eacute;s 90&amp;deg;</td><td>VWR</td><td>631-9483</td><td>Glass slides&amp;nbsp;</td></tr><tr><td>Tissue-Tek VIP 6Al</td><td>Sakura</td><td>60320417-0711 VID6E3-1</td><td>Automatic embedding device</td></tr><tr><td>Titanium forceps</td><td>Fine Science Tools</td><td>11602-16</td><td>Forceps for eggshell removal and ovarian tissue manipulation</td></tr><tr><td>Toluene, pa</td><td>VWR</td><td>28701364</td><td>Paraffin-embedding solution</td></tr></tbody></table>

the chick chorioallantoic membrane (cam) model as a tool to study ovarian tissue transplantation

Ovarian tissue cryopreservation and transplantation is an effective strategy for preserving fertility but has one major drawback, namely massive follicle loss occurring shortly after reimplantation due to abnormal follicle activation and death. Rodents are benchmark models for investigating follicle activation, but the cost, time, and ethical considerations are becoming increasingly prohibitive, thus driving the development of alternatives. The chick chorioallantoic membrane (CAM) model is particularly attractive, being inexpensive and maintaining&#160;natural immunodeficiency up to day 17 postfertilization, making it ideal to study&#160;short-term xenografting of human ovarian tissue. The CAM is also highly vascularized and has been widely used as a model to explore angiogenesis. This gives it a remarkable advantage over in vitro models and allows the investigation of mechanisms affecting the early post-grafting follicle loss process. The protocol outlined herein aims to describe the development of a CAM xenografting model for human ovarian tissue, with specific insights into the effectiveness of the technique, the graft revascularization time frame, and the tissue viability across a 6 day grafting period.

Chick embryo survival rates 
The embryo survival rate from windowing (day 3 of&#160;ED) to ovarian tissue grafting (day 7 of ED) was 79% (33/42). Since the percentage of embryonated day 0 eggs is unknown, supernumerary day 0 eggs from Lohman-selected white Leghorn chickens were ordered to ensure sufficient embryonated eggs would be available for grafting. A total of 23 viable day 7 eggs were used for grafting, one of which perished during the first 24 h, resulting in an overall embryo survival rate ...

Watch this Scientific Journal Video about The Chick Chorioallantoic Membrane CAM Model as a Tool to Study Ovarian Tissue Transplantation at JoVE.com

The Chick Chorioallantoic Membrane CAM Model as a Tool to Study Ovarian Tissue Transplantation

Here, we describe a protocol for developing a chick chorioallantoic membrane (CAM) xenografting model for human ovarian tissue and demonstrate the effectiveness of the technique, the graft revascularization time frame, and the tissue viability across a 6 day grafting period.

The Chick Chorioallantoic Membrane (CAM) Model as a Tool to Study Ovarian Tissue Transplantation

Ovarian tissue cryopreservation and transplantation is an effective strategy for preserving fertility but has one major drawback, namely massive follicle ...

the-chick-chorioallantoic-membrane-cam-model-as-tool-to-study-ovarian

Research

JoVE Journal

Biology

814 Views.  Université Catholique de Louvain. Het gebruik van CAM om vroege veranderingen na transplantatie van eierstokweefsel te observeren, heeft een aanzienlijk voordeel ten opzichte van in-vitromodellen, omdat het onderzoek mogelijk maakt naar het mechanisme dat van invloed is op het vroege vascularisatie- en follikelverliesproces. Deze techniek is goedkoop en kan de eerste 17 dagen worden uitgevoerd zonder enig risico op afstoting van het transplantaat als gevolg van de natuurlijke immunodeficiëntie van het embryo. Bovendien lever...

Video: The Chick Chorioallantoic Membrane CAM Model as a Tool to Study Ovarian Tissue Transplantation

Using the Chicken Chorioallantoic Membrane In Vivo Model to Study Gynecological and Urological Cancers

Mouse models are the benchmark tests for in vivo cancer studies. However, cost, time, and ethical considerations have led to calls for alternative in vivo cancer models. The chicken chorioallantoic membrane (CAM) model provides an inexpensive, rapid alternative that permits direct visualization of tumor development and is suitable for in vivo imaging. As such, we sought to develop an optimized protocol for engrafting gynecological and urological tumors into this model, which we present here. Approximately 7 days postfertilization, the air cell is moved to the vascularized side of the egg, where an opening is created in the shell. Tumors from murine and human cell lines and primary tissues can then be engrafted. These are typically seeded in a mixture of extracellular matrix and medium to avoid cellular dispersal and provide nutrient support until the cells recruit a vascular supply. Tumors may then grow for up to an additional 14 days prior to the eggs hatching. By implanting cells stably transduced with firefly luciferase, bioluminescence imaging can be used for the sensitive detection of tumor growth on the membrane and cancer cell spread throughout the embryo. This model can potentially be used to study tumorigenicity, invasion, metastasis, and therapeutic effectiveness. The chicken CAM model requires significantly less time and financial resources compared to traditional murine models. Because the eggs are immunocompromised and immune tolerant, tissues from any organism can potentially be implanted without costly transgenic animals (e.g., mice) required for implantation of human tissues. However, many of the advantages of this model could potentially also be limitations, including the short tumor generation time and immunocompromised/immune tolerant status. Additionally, although all tumor types presented here engraft in the chicken chorioallantoic membrane model, they do so with varying degrees of tumor growth.

We present the chicken chorioallantoic membrane model as an alternative, transplantable, in vivo model for the engraftment of gynecological and urological cancer cell lines and patient-derived tumors.

Mouse models are the benchmark tests for in vivo cancer studies. However, cost, time, and ethical considerations have led to calls for alternative in vivo ...

Cancer Research

Orthotopic Ovarian Transplantation Procedures to Investigate the Life- and Health-span Influence of Ovarian Senescence in Female Mice

Ovarian transplantation was first conducted at Utah State University in 1963. In more recent work, heterochronic transplantation of mammalian ovaries is being used to investigate the health-protective effects of young ovaries in young females. The current procedures employ an orthotopic transplantation method, where allogenic ovaries are transplanted back to their original position in the ovarian bursa. This is in contrast to the more commonly used heterotopic transplantation of ovaries/ovarian tissue subcutaneously or under the kidney capsule. All three locations provide efficient revascularization of the transplanted tissues. However, orthotopic transplantation provides the ovary with the most natural signaling environment and is the only procedure that provides the opportunity for the animal to reproduce naturally post-operatively. One must take care to remove all endogenous ovarian tissue during the ovariectomy procedure. If any endogenous tissue remains or if only one ovary is removed, the transplanted tissue will remain dormant until the existing tissue becomes senescent. While revascularization of the transplanted ovaries occurs very quickly, the transplant recipient can take a considerable amount of time to adapt to a new hypothalamic/pituitary/gonadal/adrenal (HPG/A) axis signaling regime associated with the transplanted tissue. This normally takes about 100 days in the mouse. Therefore, transplantation experiments should be designed to accommodate this adaptation period. Typical results with ovarian transplantation will include changes in the health of the recipient that reflect the age of the transplanted ovary, rather than the chronological age of the recipient.

Described here are orthotopic ovarian transplantation protocols for modifying ovarian influence on the physiology of the recipient mouse. This surgical procedure is used to expose female mice to various types of ovarian influence and may include heterochronic transplantations and transplantation of ovaries that have undergone various ex vivo treatments.

Ovarian transplantation was first conducted at Utah State University in 1963. In more recent work, heterochronic transplantation of mammalian ovaries is ...

Developmental Biology

The In ovo CAM-assay as a Xenograft Model for Sarcoma

Sarcoma is a very rare disease that is heterogeneous in nature, all hampering the development of new therapies. Sarcoma patients are ideal candidates for personalized medicine after stratification, explaining the current interest in developing a reproducible and low-cost xenotransplant model for this disease. The chick chorioallantoic membrane is a natural immunodeficient host capable of sustaining grafted tissues and cells without species-specific restrictions. In addition, it is easily accessed, manipulated and imaged using optical and fluorescence stereomicroscopy. Histology further allows detailed analysis of heterotypic cellular interactions.
This protocol describes in detail the in ovo grafting of the chorioallantoic membrane with fresh sarcoma-derived tumor tissues, their single cell suspensions, and permanent and transient fluorescently labeled established sarcoma cell lines (Saos-2 and SW1353). The chick survival rates are up to 75%. The model is used to study graft- (viability, Ki67 proliferation index, necrosis, infiltration) and host (fibroblast infiltration, vascular ingrowth) behavior. For localized grafting of single cell suspensions, ECM gel provides significant advantages over inert containment materials. The Ki67 proliferation index is related to the distance of the cells from the surface of the CAM and the duration of application on the CAM, the latter determining a time frame for the addition of therapeutic products.

The In ovo CAM-assay as a Xenograft Model for Sarcoma

The in ovo chorioallantoic membrane (CAM) is grafted with fresh sarcoma-derived tumor tissues, their single cell suspensions, and permanent and transient fluorescently labeled established sarcoma cell lines. The model is used to study graft- (viability, Ki67 proliferation index, necrosis, infiltration) and host (fibroblast infiltration, vascular ingrowth) behavior.

Sarcoma is a very rare disease that is heterogeneous in nature, all hampering the development of new therapies. Sarcoma patients are ideal candidates for ...

Medicine

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.

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.

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 ...

Establishing a 3D Ex Vivo Model for Ovarian Cancer-Omentum Interaction

An Ex Vivo Model of Ovarian Cancer Peritoneal Metastasis Using Human Omentum

Ovarian cancer is the deadliest gynecologic malignancy. The omentum plays a key role in providing a supportive microenvironment to metastatic ovarian cancer cells as well as immune modulatory signals that allow tumor tolerance. However, we have limited models that closely mimic the interaction between ovarian cancer cells and adipose-rich tissues. To further understand the cellular and molecular mechanisms by which the omentum provides a pro-tumoral microenvironment, we developed a unique 3D ex vivo model of cancer cell-omentum interaction. Using human omentum, we are able to grow ovarian cancer cells within this adipose-rich microenvironment and monitor the factors responsible for tumor growth and immune regulation. In addition to providing a platform for the study of this adipose-rich tumor microenvironment, the model provides an excellent platform for the development and evaluation of novel therapeutic approaches to target metastatic cancer cells in this niche. The proposed model is easy to generate, inexpensive, and applicable to translational investigations.

Author Spotlight: Advanced Ex Vivo Model for Investigating Cancer-Adipose Microenvironment Interaction 

This protocol describes the establishment of a three-dimensional (3D) ex vivo model of cancer cell-omentum interaction. The model provides a platform for elucidating pro-tumor mechanisms within the adipose niche and for testing novel therapies.

Ovarian cancer is the deadliest gynecologic malignancy. The omentum plays a key role in providing a supportive microenvironment to metastatic ovarian ...

The In Ovo Chick Chorioallantoic Membrane (CAM) Assay as an Efficient Xenograft Model of Hepatocellular Carcinoma

The chick chorioallantoic membrane (CAM) begins to develop by day 7 after fertilization and matures by day 12. The CAM is naturally immunodeficient and highly vascularized, making it an ideal system for tumor implantation. Furthermore, the CAM contains extracellular matrix proteins such as fibronectin, laminin, collagen, integrin alpha(v)beta3, and MMP-2, making it an attractive model to study tumor invasion and metastasis. Scientists have long taken advantage of the physiology of the CAM by using it as a model of angiogenesis. More recently, the CAM assay has been modified to work as an in vivo xenograft model system for various cancers that bridges the gap between basic in vitro work and more complex animal cancer models. The CAM assay allows for the study of tumor growth, anti-tumor therapies, and pro-tumor molecular pathways in a biologically relevant system that is both cost- and time-effective. Here, we describe the development of CAM xenograft model of hepatocellular carcinoma (HCC) with embryonic survival rates of up to 93% and reliable tumor take leading to growth of three-dimensional, vascularized tumors.

The In Ovo Chick Chorioallantoic Membrane CAM Assay as an Efficient Xenograft Model of Hepatocellular Carcinoma

The chick chorioallantoic membrane (CAM) is immunodeficient and highly vascularized, making it a natural in vivo model of tumor growth and angiogenesis. In this protocol, we describe a reliable method of growing three-dimensional, vascularized hepatocellular carcinoma (HCC) tumors using the CAM assay.

The chick chorioallantoic membrane (CAM) begins to develop by day 7 after fertilization and matures by day 12. The CAM is naturally immunodeficient and ...

Transplantation Into the Mouse Ovarian Fat Pad

Orthotopic transplantation assays in mice are invaluable for studies of cell regeneration and neoplastic transformation. Common approaches for orthotopic transplantation of ovarian surface and tubal epithelia include intraperitoneal and intrabursal administration of cells. The respective limitations of these methods include poorly defined location of injected cells and limited space volume. Furthermore, they are poorly suited for long-term structural preservation of transplanted organs. To address these challenges, we have developed an alternative approach, which is based on the introduction of cells and tissue fragments into the mouse fat pad. The mouse ovarian fat pad is located in the immediate vicinity of the ovary and uterine tube (aka oviduct, fallopian tube), and provides a familiar microenvironment for cells and tissues of these organs. In our approach fluorescence-labeled mouse and human cells, and fragments of the uterine tube are engrafted by using minimally traumatic dorsal incision surgery. Transplanted cells and their outgrowths are easily located in the ovarian fat pad for over 40 days. Long-term transplantation of the entire uterine tube allows correct preservation of all principle tissue components, and does not result in adverse side effects, such as fibrosis and inflammation. Our approach should be uniquely applicable for answering important biological questions such as differentiation, regenerative and neoplastic potential of specific cell populations. Furthermore, it should be suitable for studies of microenvironmental factors in normal development and cancer.

We describe an ovarian fad pad transplantation assay suitable for studies of normal and transformed epithelia of the female reproductive tract. The mouse fat pad allows transplantation of large tissue fragments, is easily accessible for surgery and imaging, and provides the most favorable native environment for tissues of M&#252;llerian origin.

Orthotopic transplantation assays in mice are invaluable for studies of cell regeneration and neoplastic transformation. Common approaches for orthotopic ...

The Chick Chorioallantoic Membrane In Vivo Model to Assess Perineural Invasion in Head and Neck Cancer

Perineural invasion is a phenotype in which cancer surrounds or invades the nerves. It is associated with poor clinical outcome for head and neck squamous cell carcinoma and other cancers. Mechanistic studies have shown that the molecular crosstalk between nerves and tumor cells occurs prior to physical interaction. There are only a few in vivo models to study perineural invasion, especially to investigate early progression, before physical nerve-tumor interactions occur. The chick chorioallantoic membrane model has been used to study cancer invasion, because the basement membrane of the chorionic epithelium mimics that of human epithelial tissue. Here we repurposed the chick chorioallantoic membrane model to investigate perineural invasion, grafting rat dorsal root ganglia and human head and neck squamous cell carcinoma cells onto the chorionic epithelium. We have demonstrated how this model can be useful to evaluate the ability of cancer cells to invade neural tissue in vivo.&#160;

The Chick Chorioallantoic Membrane In Vivo Model to Assess Perineural Invasion in Head and Neck Cancer

Perineural invasion is an aggressive phenotype for head and neck squamous cell carcinomas and other tumors. The chick chorioallantoic membrane model has been used for studying angiogenesis, cancer invasion, and metastasis. Here we demonstrate how this model can be utilized to assess perineural invasion in vivo.

Perineural invasion is a phenotype in which cancer surrounds or invades the nerves. It is associated with poor clinical outcome for head and neck squamous ...

Chicken Chorioallantoic Membrane-based Cancer Modeling: An In Ovo Model to Study Cancer Cell Tumorigenesis and Metastasis

Source: Sharrow, A. C. et al. <a href="https://www.jove.com/t/60651">Using the Chicken Chorioallantoic Membrane In Vivo Model to Study Gynecological and Urological Cancers.</a> J. Vis. Exp.&#160;(2020)
In this video, we describe the implantation of cancer cells onto a chicken chorioallantoic membrane to generate an in ovo model. The model successfully engrafts the ovarian cancer cells to study the progression of gynecological cancer.

Source: Sharrow, A. C. et al. Using the Chicken Chorioallantoic Membrane In Vivo Model to Study Gynecological and Urological Cancers. J. Vis. ...

JoVE Encyclopedia of Experiments

Gynecologic Cancer

Animal model studies

Chick Chorioallantoic Membrane or CAM Tumor Model: An In Ovo Method to Simulate Perineural Invasion of Tumor Cells

Source: Schmidt, L. B. et al. <a href="https://www.jove.com/video/59296" target="_blank">The Chick Chorioallantoic Membrane In Vivo Model to Assess Perineural Invasion in Head and Neck Cancer.</a> J. Vis. Exp. (2019)
In this video, we describe the method to co-culture dorsal root ganglia and cancer cells on chick chorioallantoic membrane or CAM to study perineural invasion.

Source: Schmidt, L. B. et al. The Chick Chorioallantoic Membrane In Vivo Model to Assess Perineural Invasion in Head and Neck Cancer. J. Vis. Exp. (2019)
...

The Chick Chorioallantoic Membrane (CAM) Model as a Tool to Study Ovarian Tissue Transplantation

Samenvatting

Meer video's verkennen