Name: quail chorioallantoic membrane - a tool for photodynamic diagnosis and therapy
Uploaded: 2022-04-28T12:30:00.000+00:00
Duration: 7 min 43 s
Description: Watch this Scientific Journal Video about Quail Chorioallantoic Membrane - A Tool for Photodynamic Diagnosis and Therapy at JoVE.com

The work was supported by VEGA 2/0042/21 and APVV 20-0129. The contribution of V. Hunto&#353;ov&#225; is the result of the project implementation: Open scientific community for modern interdisciplinary research in medicine (Acronym: OPENMED), ITMS2014+: 313011V455 supported by the Operational Program Integrated Infrastructure, funded by the ERDF.

The research was performed in compliance with institutional guidelines. All equipment and reagents must be autoclaved or sterilized with 70% ethanol or UV light.
1. Egg incubation
<ol>
	<li>Store fertilized quail eggs at 10-15 &#176;C for a maximum of 4-5 days before starting incubation. Use only clean and undamaged eggs.</li>
	<li>Incubate the eggs in a forced draught incubator for ~ 53-54 h. Lay the eggs horizontally with the egg rotation switched off, at 50%-60% humidity and 37.5 &#176;C incubation temperature.</li>
</ol>
2. Ex ovo culture preparation
NOTE: After initial incubation, the eggs are suitable for starting the ex ovo cultivation.
<ol>
	<li>Disinfect the egg surface with 70% ethanol, without rotating the egg.</li>
	<li>In a sterile laminar-flow cabinet, open the eggshell using small sterile surgical scissors and transfer the contents into a 6-well culture plate. If properly done, the embryo will lie on top of an undamaged yolk. After each egg, disinfect the scissors with 70% ethanol.</li>
	<li>Add approximately 5 mL of sterile water to the gaps in the 6-well plate, as humidity is essential to prevent the CAM from drying out.</li>
	<li>Place the embryos in an incubator until further experiments, while maintaining a temperature of 37 &#176;C and 80%-90% humidity.</li>
	<li>When the CAM is fully developed (from ED7), place a sterilized silicone ring (6 mm in diameter and approximately 1.5 mm in thickness) on the CAM surface, along the small capillaries. Avoid major blood vessels when placing the ring. 
	NOTE: The ring defines the workspace, helps mark the place of substance application, and prevents the liquid contents from leaking out.</li>
	<li>Terminate the cultivation of the embryos according to the country&#39;s legislation.</li>
	<li>Importantly, wear gloves or keep hands disinfected with 70% ethanol during work. Aspirate improperly tipped embryos or unfertilized eggs with a vacuum aspirator.</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63422/63422fig01.jpg" /> 
Figure 1: Ex ovo culture preparation. (A) Japanese quail eggs as they are stored and incubated. (B) Egg surface disinfection with ethanol. (C) Eggshell is cut with scissors. (D) The content of the egg is emptied into the well. (E) Properly prepared 3-day old embryo, with developing CAM vasculature. (F) Culture plates stored in an incubator. <a href="https://www.jove.com/files/ftp_upload/63422/63422fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63422/63422fig02.jpg" /> 
Figure 2: 6-well culture plate with embryos and silicone rings placed on top. <a href="https://www.jove.com/files/ftp_upload/63422/63422fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
3. Inoculation of tumor cells
NOTE: All procedures require the use of a sterile laminar flow cabinet.
<ol>
	<li>Culture different types of adherent cells in flasks according to respective culture protocols.
	<ol>
		<li>Remove the old medium from the flasks and rinse the cell layer with sterile phosphate-buffered saline (PBS) to remove traces of the medium.</li>
		<li>After trypsinization, inhibit the trypsin by adding the culture medium and harvest the cells into centrifuge tubes. Centrifuge the cells and resuspend the cell pellet in a fresh culture medium. Count the cells and resuspend them in a cell culture medium at the desired concentration. 
		NOTE: It is also possible to produce 3D cell cultures, i.e., spheroids using, for example, the hanging drop method21.</li>
	</ol>
	</li>
	<li>Implant 1 x 105-1 x 106 tumor cells or 5-15 spheroids (per CAM) in 30 &#181;L of cell medium solution into the silicone ring on top of the CAM. 
	NOTE: The volume depends on the size of the silicone ring used. If a silicone ring with a larger diameter is used, a larger volume is needed to cover the entire area. Depending on the cell type, or for different types of experiments, various cell concentrations may be used. Sometimes, fine scraping of CAM is used to improve the adhesion of the embedded cells.</li>
	<li>Return the CAMs with inoculated cells to the incubator (37 &#176;C and 80%-90% humidity).</li>
</ol>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63422/63422fig03.jpg" /> 
Figure 3: Inoculation of CAM with tumors. (A) Aspiration of spheroids with a pipette, and (B) implantation on the CAM surface. <a href="https://www.jove.com/files/ftp_upload/63422/63422fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
4. Application of photosensitizer
<ol>
	<li>Application of hypericin
	<ol>
		<li>Under dim lighting, prepare a 2 mM stock solution of hypericin by dissolving it in 100% dimethyl sulfoxide (DMSO). Prepare a 79 &#181;M working solution of hypericin shortly before the experiment with sterile PBS or saline solution. Ensure the final concentration of DMSO in all solutions is always under 0.2%, which does not affect the development of the embryo.</li>
		<li>Under sterile conditions, apply an appropriate volume of hypericin solution to the silicone ring. A volume of 30 &#181;L is sufficient to fill a ring with a diameter of 6 mm.</li>
		<li>Keep embryos in an incubator at 37 &#176;C and 80%-90% humidity. Hypericin in an aqueous solution is photoactive after its monomerization in the tissue, approximately 3 h after its application on the CAM.</li>
	</ol>
	</li>
	<li>Application of&#160;hypericin with LDL, HDL, or nanoparticles as transport systems 
	NOTE: These transport systems are used to improve the penetration of hypericin into cells and cell structures.
	<ol>
		<li>Due to the photosensitivity, perform all the steps, including the preparation of solutions, under dim lighting and store the solutions in the dark.</li>
		<li>Prepare the hypericin-LDL and hypericin-HDL formulations by mixing appropriate volumes of lipoproteins and hypericin stock solutions in PBS according to reference15. The concentration of LDL or HDL to hypericin is 20:115.</li>
		<li>Synthesize polymer nanoparticles by living cationic ring-opening polymerization according to references12,13. Adjust the concentration of hypericin in polymer nanoparticles with PBS to 10&#160;&#956;M12,13.</li>
	</ol>
	</li>
</ol>
5. PDD and PDT
<ol>
	<li>Conduct PDD
	<ol>
		<li>Under sterile conditions, apply photosensitizer (hypericin, hypericin-loaded polymeric nanoparticles, or hypericin with lipoprotein carrier) on the CAM surface, with or without tumor cells (ED 7-9).</li>
		<li>Illuminate the CAM using violet excitation light (custom-made circular LED light of 405 nm wavelength) and record the fluorescence of hypericin in CAM tissue and tumor cells with a digital camera at time intervals of 0, 1, 3, 5, 24, and 48 h after hypericin administration.</li>
		<li>If the research requires an image of the CAM in white light, record the CAM before hypericin administration and at the end of the experiment, shortly before the tissue fixation, as the light affects the photoactivation of hypericin.</li>
		<li>Evaluate the fluorescence intensity using an image processing and analysis program (e.g., ImageJ22).
		<ol>
			<li>Split RGB image channels into separate red, green, and blue images. Analyze the red intensity image in 8-bit form. Evaluate the red intensity from the area inside the ring and approximate it as the hypericin fluorescence. Obtain whole image profile plots (using ImageJ&#39;s Profile Plot plugin) at different time intervals after hypericin administration (i.e., from 0 h, right after administration, up to 48 h).</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Conduct PDT
	<ol>
		<li>Perform the PDT at least 3 h after hypericin application.</li>
		<li>Place the optical fiber above the surface of the CAM for the laser ray to cover the entire area within the silicone ring.</li>
		<li>Perform in vivo irradiation (1-2 min) with a 405 nm laser light at a fluence rate of 285 mW/cm2.</li>
		<li>Record CAM using white light and 405 nm fluorescent light before and after photodynamic treatment (24 h and 48 h).</li>
		<li>Detect photodamage 24 h and 48 h after PDT from the images taken in white light. Evaluate vascular damage by a semiquantitative score16: 1&#160;-&#160;no&#160;destruction;&#160;2&#160;-&#160;partial closure of capillaries (diameter &#8804;10 &#181;m) with no destruction of medium or larger (diameter &#8805;50 &#181;m) and smaller vessels (diameter 10-50 &#181;m); 3 - partial closure of smaller vessels with capillaries vanishing; 4 - partial closure of larger vessels with smaller vessels and capillaries vanishing; and 5 - total photo thrombosis with most of the vessels vanishing.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/63422/63422fig04.jpg" /> 
Figure 4: Treatment of CAM with laser light. This picture was taken for illustrative purposes. For PDD or PDT, the room must be dark. <a href="https://www.jove.com/files/ftp_upload/63422/63422fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
6. Preparing CAM for further evaluation
<ol>
	<li>Paraffin embedding
	<ol>
		<li>Fix the CAM tissue in a cultivation plate with 4% paraformaldehyde (PFA) in PBS for a minimum of 2&#160;h and a maximum of overnight.</li>
		<li>Remove the PFA and carefully cut out from the CAM a part of the tissue within the silicone ring.</li>
		<li>Immediately dehydrate the CAM tissue in ascending alcohol series as follows. Place the CAM tissue in 70% ethanol for 3 min, eosin solution for 2 min (for easier location of the tissue in the paraffin block), 96% ethanol for 3 x 5 min (always in a new Petri dish), and 100% ethanol for 5 min and xylene for 2 x 10 min.</li>
		<li>Using a spatula or thin brush, transfer the samples as quickly as possible to the dissolved paraffin in Petri dishes (59 &#176;C). After 24 h, place the tissue in histological mold, fill it with paraffin embedding medium, and allow it to solidify in the refrigerator. Cut the solidified CAM from the embedding medium, turn it in the tray by 90&#176;, fill it again with the embedding medium, and let it solidify.</li>
		<li>Prepare 5-10 &#181;m sections on a microtome for histopathological analysis to determine PDT-induced damage.</li>
	</ol>
	</li>
	<li>Preparation of frozen CAM sections for histology 
	NOTE: For some methodologies including histology, frozen sections prepared on cryostat microtome are more suitable. Follow the below steps for preparing the frozen CAM sections.
	<ol>
		<li>Carefully mount native or 4% paraformaldehyde-fixed CAM on the glass slide.</li>
		<li>Fill embedding mold to half with optimal cutting temperature compound (OCT) and freeze in liquid nitrogen or a mixture of dry ice and ethanol.</li>
		<li>After freezing, carefully tilt and slide the CAM from the glass slide onto the top of the frozen OCT medium. Place again in the mold, cover with OCT medium, and freeze as in Step 6.2.2.</li>
	</ol>
	</li>
	<li>Fractal analysis of quail CAM 
	NOTE: Vasculature changes caused by PDT can be assessed by calculating the fractal dimension coefficient19.
	<ol>
		<li>In the laminar flow cabinet, overflow CAM in a cultivation plate with a pre-warmed (37 &#176;C) fixation solution of 4% paraformaldehyde and 2% glutaraldehyde in PBS.</li>
		<li>Remove the fixation solution after 48 h. Carefully separate the CAM from the embryo with micro-scissors and a fine brush and wash it in PBS.</li>
		<li>Mount the washed CAM on a glass slide and let it slowly dry.</li>
		<li>Photograph the slide using a digital camera and a transilluminator as a source of homogenous white light.</li>
		<li>Process digital images using ImageJ software22. For the analysis, select a square region (512 &#215; 512 pixels) from the area with distal arterial branches. Binarize and skeletonize the images and calculate the fractal dimension coefficient (Df) following the procedures described in reference31.</li>
	</ol>
	</li>
	<li>Molecular analysis of CAM tissue
	<ol>
		<li>For molecular analysis, carefully separate native CAM, freeze in liquid nitrogen, and store at -80 &#176;C.</li>
		<li>Determine gene expression according to standard protocols for RNA isolation23, reverse transcription into cDNA, and quantitative PCR24.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/63422/63422fig05.jpg" /> 
Figure 5: CAM tissue for fractal analysis. After PDT, CAM is fixed, mounted on a slide, and dried for fractal analysis. The picture was taken in white light using a transilluminator. <a href="https://www.jove.com/files/ftp_upload/63422/63422fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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A., 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=Extending+the+applicability+of+in+ovo+and+ex+ovo+chicken+chorioallantoic+membrane+assays+to+study+cytostatic+activity+in+neuroblastoma+cells.">Extending the applicability of in ovo and ex ovo chicken chorioallantoic membrane assays to study cytostatic activity in neuroblastoma cells.</a> Frontiers in Oncology. 11, 1-10 (2021).</li><li>Meta, M., Kundekov&#225;, B., Bil&#269;&#237;k, B., M&#225;&#269;ajov&#225;, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+silicone+ring+application+on+CAM+vasculature+in+Japanese+Quail+(Coturnix+japonica).">The effect of silicone ring application on CAM vasculature in Japanese Quail (Coturnix japonica).</a> Proceedings of the Student Scientific Conference Faculty of Natural Sciences of Comenius University, Bratislava, Slovakia. , 385-390 (2019).</li><li>Kohli, N., 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=Pre-screening+the+intrinsic+angiogenic+capacity+of+biomaterials+in+an+optimised+ex+ovo+chorioallantoic+membrane+model.">Pre-screening the intrinsic angiogenic capacity of biomaterials in an optimised ex ovo chorioallantoic membrane model.</a> Journal of Tissue Engineering. 11, 1-15 (2020).</li><li>Kundekov&#225;, B., M&#225;&#269;ajov&#225;, M., Meta, M., &#268;avarga, I., Bil&#269;&#237;k, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chorioallantoic+membrane+models+of+various+avian+species+differences+and+applications.">Chorioallantoic membrane models of various avian species differences and applications.</a> Biology-Basel. 10 (4), 301 (2021).</li><li>Parsons-Wingerter, P., Elliott, K. E., Clark, J. I., Farr, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroblast+growth+factor-2+selectively+stimulates+angiogenesis+of+small+vessels+in+arterial+tree.">Fibroblast growth factor-2 selectively stimulates angiogenesis of small vessels in arterial tree.</a> Arteriosclerosis, Thrombosis and Vascular Biology. 20 (5), 1250-1256 (2000).</li><li>Buzz&#225;, H. H., Zangirolami, A. C., Davis, A., G&#243;mez-Garc&#237;a, P. B., Kurachi, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+analysis+of+a+tumor+model+in+the+chorioallantoic+membrane+used+for+the+evaluation+of+different+photosensitizers+for+photodynamic+therapy.">Fluorescence analysis of a tumor model in the chorioallantoic membrane used for the evaluation of different photosensitizers for photodynamic therapy.</a> Photodiagnosis and Photodynamic Therapy. 19, 78-83 (2017).</li></ol>

The authors have no conflicts of interest.

For successful ex ovo cultivation, it is important to follow the protocol above. Moreover, if the eggs are not opened carefully enough or there is insufficient humidity during the cultivation, the yolk sack sticks to the shell and often ruptures. The start of an ex ovo cultivation at the time of about 60 h of egg incubation ensures the high survival rate of the embryos, as they are already large enough to survive the handling. At the later developmental stages, the CAM becomes thinner and adheres to the...

The chicken chorioallantoic membrane (CAM) model is well known and widely used in various areas of research. It is a richly vascularized extraembryonic organ that provides gas exchange and mineral transport1. Due to the transparency and accessibility of this membrane, individual blood vessels and their structural changes can be observed in real time2. Despite the advantages, chick CAM also has some limitations (e.g., larger breeding facilities, egg production, and feed consumption) that could be avoided by using other avian species. In this protocol, an alternative ex ovo CAM model using Japanese quail (Coturnix japonica) embryo is described. Due to its small size, it allows the use of a much larger number of experimental individuals than chicken CAM. Moreover, the shorter 16-day embryonic development of quail embryos is another advantage. The first larger vessels on quail CAM appear on embryonic day (ED) 7. This can be directly compared with chick embryo development (stages 4-35); however, the later stages of development are no longer comparable and require less time for the quail embryo3. Of interest is the regular occurrence of microvascular branching similar to that of chicken CAMs4,5,6. Rapid sexual maturation, high egg production, and low-cost breeding are other examples that favor the use of this experimental model7.
An avian CAM model is often used in photodynamic therapy (PDT) studies8. PDT is used to treat several forms of cancer (small localized tumors) and other non-oncological diseases. Its principle is in the delivery of a fluorescent drug, a photosensitizer (PS), to the damaged tissue and its activation with light of the appropriate wavelength. One prospective PS used in research is hypericin, originally isolated from the medicinal plant St. John&#39;s wort (Hypericum perforatum)9. The strong photosensitizing effects of this compound are based on its photochemical and photophysical properties. These are characterized by multiple fluorescence excitation peaks in the 400-600 nm range, which induce the emission of fluorescence at about 600 nm. The absorption maxima of hypericin within the spectral band are in the 540-590 nm range, and the fluorescence maxima are in the 590-640 nm range9. To achieve these photosensitizing effects, hypericin is excited by laser light at a wavelength of 405 nm after local administration10. In the presence of light, hypericin can exhibit virucidal, antiproliferative, and cytotoxic effects11, while there is no systemic toxicity, and it is rapidly released from the organism. Hypericin is a lipophilic substance that forms water-insoluble, non-fluorescent aggregates, which is why several types of nanocarriers, such as polymeric nanoparticles12,13 or high- and low-density lipoproteins (HDL, LDL)14,15, are used to help its delivery and penetration into the cells. Since CAM is a naturally immunodeficient system, tumor cells can be implanted directly on the membrane surface. The model is also well suited for recording the extent of PDT-induced vascular damage according to a defined score16,17. Light of lower intensity compared to PDT can be used for photodynamic diagnosis (PDD). Monitoring the tissue under violet excitation LED light also leads to photoactivation of photosensitizers18,19,20 that results in an emission of fluorescent light, yet it does not provide enough energy to start a PDT reaction and damage the cells. It makes it a good tool for tumor visualization and diagnosis or monitoring the pharmacokinetics of used PSs14,15.
This article describes the preparation of the quail ex ovo CAM assay with survival rates over 80%. This ex ovo culture was successfully applied in a large number of experiments.

<table><tbody><tr><td>6-Well Cell Culture Plate</td><td>Sarstedt</td><td>83.392</td><td>Transparent polystyrene, sterile</td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; Incubator ESCO CCL-0508</td><td>ESCO, Singapore</td><td>CCL-050B-8</td><td>CO&lt;sub&gt;2&lt;/sub&gt; cell culture incubator</td></tr><tr><td>cryocut Leica CM 1800</td><td>Reichert-Jung, USA</td><td></td><td></td></tr><tr><td>digital camera Canon EOS 6D II</td><td>Canon, Japan</td><td></td><td></td></tr><tr><td>diode laser 405 nm</td><td>Ocean Optics, USA</td><td></td><td></td></tr><tr><td>DMSO</td><td>Sigma-Aldrich</td><td>67-68-5</td><td>dimethyl sulfoxid</td></tr><tr><td>eosin</td><td>Sigma-Aldrich</td><td>15086-94-9</td><td></td></tr><tr><td>ethanol</td><td>Sigma-Aldrich</td><td>64-17-5</td><td></td></tr><tr><td>fine brush size 2</td><td>Faber-Castell</td><td>281802</td><td>brush for CAM separation and manipulation</td></tr><tr><td>glutaraldehyde</td><td>Sigma-Aldrich</td><td>111-30-8</td><td></td></tr><tr><td>hematoxylin</td><td>Sigma-Aldrich</td><td>517-28-2</td><td></td></tr><tr><td>hypericin</td><td>Sigma-Aldrich</td><td>84082-80-4</td><td></td></tr><tr><td>incubator Bios Midi</td><td>Bios Sedl&lt;img alt=&quot;Equation 1&quot; src=&quot;/files/ftp_upload/63422/63422eq01.jpg&quot; style=&quot;margin: auto;&quot;/&gt;any, Czech Republic</td><td></td><td>Forced draught incubator for initial incubation</td></tr><tr><td>incubator Memmert IF160</td><td>Memmert, Germany</td><td></td><td>Forced air circulation incubator for CAM incubation</td></tr><tr><td>Kaiser slimlite plano, LED light box</td><td>Kaiser, Germany</td><td>2453</td><td>Transilluminator</td></tr><tr><td>LED light 405 nm</td><td></td><td></td><td>custom made circular LED light</td></tr><tr><td>macro lens Canon MP- E 65 mm f/2.8</td><td>Canon, Japan</td><td></td><td></td></tr><tr><td>microscope Kapa 2000</td><td>Kvant, Slovakia</td><td></td><td>optical microscope</td></tr><tr><td>microtome Auxilab 508</td><td>Auxilab, Spain</td><td></td><td>manual rotary microtome</td></tr><tr><td>paraformaldehyde</td><td>Sigma-Aldrich</td><td>30525-89-4</td><td></td></tr><tr><td>Paraplast Plus</td><td>Sigma-Aldrich</td><td>P3683</td><td>parafin medium for tissue embedding</td></tr><tr><td>PBS</td><td>Sigma-Aldrich</td><td>P4417</td><td>Phosphate saline buffer</td></tr><tr><td>scissors Castroviejo</td><td>Orimed</td><td>&amp;nbsp;OR66-108</td><td>micro scissors for CAM separation</td></tr><tr><td>software ImageJ 1.53</td><td>public domain</td><td></td><td>image processing and analysis program</td></tr><tr><td>stock solution HDL</td><td>Sigma-Aldrich</td><td>437641-10MG</td><td>high density lipoproteins</td></tr><tr><td>stock solution LDL</td><td>Sigma-Aldrich</td><td>437644-10MG</td><td>low density lipoproteins</td></tr><tr><td>Tissue-Tek O.C.T. Compound</td><td>Sakura Finetek</td><td>4583</td><td>Optimal Cutting Temperature Compound 118 mL squeeze bottles</td></tr></tbody></table>

quail chorioallantoic membrane - a tool for photodynamic diagnosis and therapy

The chorioallantoic membrane (CAM) of an avian embryo is a thin, extraembryonic membrane that functions as a primary respiratory organ. Its properties make it an excellent in vivo experimental model to study angiogenesis, tumor growth, drug delivery systems, or photodynamic diagnosis (PDD) and photodynamic therapy (PDT). At the same time, this model addresses the requirement for the replacement of experimental animals with a suitable alternative. Ex ovo cultivated embryo allows easy substance application, access, monitoring, and documentation. The most frequently used is chick CAM; however, this article describes the advantages of the Japanese quail CAM as a low-cost and high-throughput model. Another advantage is the shorter embryonic development, which allows higher experimental turnover. The suitability of quail CAM for PDD and PDT of cancer and microbial infections is explored here. As an example, the use of the photosensitizer hypericin in combination with lipoproteins or nanoparticles as a delivery system is described. The damage score from images in white light and changes in fluorescence intensity of the CAM tissue under violet light (405 nm) was determined, together with analysis of histological sections. The quail CAM clearly showed the effect of PDT on the vasculature and tissue. Moreover, changes like capillary hemorrhage, thrombosis, lysis of small vessels, and bleeding of larger vessels could be observed. Japanese quail CAM is a promising in vivo model for photodynamic diagnosis and therapy research, with applications in studies of tumor angiogenesis, as well as antivascular and antimicrobial therapy.

The localization&#160;of the tumor on the CAM surface is difficult in white light. Photosensitizer (here, hypericin) used in PDD is expected to be taken up selectively by the tumor and helps visualize the tumor. The addition of hypericin and the use of fluorescent light (e.g., 405 nm) showed the tumor (squamous cell carcinoma TE1) position very well (Figure 6A). Histological analysis showed vital tumor cells invading healthy tissues. Concentric structures of abnormal squamous cells, often de...

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Quail Chorioallantoic Membrane - A Tool for Photodynamic Diagnosis and Therapy

The chorioallantoic membrane (CAM) of the avian embryo is a very useful and applicable tool for various areas of research. A special ex ovo model of Japanese quail CAM is suitable for photodynamic treatment investigation.

The chorioallantoic membrane (CAM) of an avian embryo is a thin, extraembryonic membrane that functions as a primary respiratory organ. Its properties ...

quail-chorioallantoic-membrane-tool-for-photodynamic-diagnosis

Research

JoVE Journal

Cancer Research

2.6K Views.  IABG, Bratislava, Slovakia. The chorioallantoic membrane (CAM) of the avian embryo is a very useful and applicable tool for various areas of research. A special ex ovo model of Japanese quail CAM is suitable for photodynamic treatment investigation.

Video: Quail Chorioallantoic Membrane - A Tool for Photodynamic Diagnosis and Therapy

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 ...

Novel Photoacoustic Microscopy and Optical Coherence Tomography Dual-modality Chorioretinal Imaging in Living Rabbit Eyes

Photoacoustic ocular imaging is an emerging ophthalmic imaging technology that can noninvasively visualize ocular tissue by converting light energy into sound waves and is currently under intensive investigation. However, most reported work to date is focused on the imaging of the posterior segment of the eyes of small animals, such as rats and mice, which poses challenges for clinical human translation due to small eyeball sizes. This manuscript describes a novel photoacoustic microscopy (PAM) and optical coherence tomography (OCT) dual-modality system for posterior segment imaging of the eyes of larger animals, such as rabbits. The system configuration, system alignment, animal preparation, and dual-modality experimental protocols for in vivo, noninvasive, label-free chorioretinal imaging in rabbits are detailed. The effectiveness of the method is demonstrated through representative experimental results, including retinal and choroidal vasculature obtained by the PAM and OCT. This manuscript provides a practical guide to reproducing the imaging results in rabbits and advancing photoacoustic ocular imaging in larger animals.

This manuscript describes the novel setup and operating procedure of a photoacoustic microscopy and optical coherence tomography dual-modality system for noninvasive, label-free chorioretinal imaging of larger animals, such as rabbits.

Photoacoustic ocular imaging is an emerging ophthalmic imaging technology that can noninvasively visualize ocular tissue by converting light energy into ...

Engineering

Discovery of Metastatic Regulators using a Rapid and Quantitative Intravital Chick Chorioallantoic Membrane Model

Recent advances in cancer research has illustrated the highly complex nature of cancer metastasis. Multiple genes or genes networks have been found to be involved in differentially regulating cancer metastatic cascade genes and gene products dependent on the cancer type, tissue, and individual patient characteristics. These represent potentially important targets for genetic therapeutics and personalized medicine approaches. The development of rapid screening platforms is essential for the identification of these genetic targets.
The chick chorioallantoic membrane (CAM) is a highly vascularized, collagen rich membrane located under the eggshell that allows for gas exchange in the developing embryo. Due to the location and vascularization of the CAM, we developed it as an intravital human cancer metastasis model that allows for robust human cancer cell xenografting and real-time imaging of cancer cell interactions with the collagen rich matrix and vasculature. 
Using this model, a quantitative screening platform was designed for the identification of novel drivers or suppressors of cancer metastasis. We transduced a pool of head and neck HEp3 cancer cells with a complete human genome shRNA gene library, then injected the cells, at low density, into the CAM vasculature. The cells proliferated and formed single-tumor cell colonies. Individual colonies that were unable to invade into the CAM tissue were visible as a compact colony phenotype and excised for identification of the transduced shRNA present in the cells. Images of individual colonies were evaluated for their invasiveness. Multiple rounds of selections were performed to decreases the rate of false positives. Individual, isolated cancer cell clones or newly engineered clones that express genes of interest were subjected to primary tumor formation assay or cancer cell vasculature co-option analysis. In summary we present a rapid screening platform that allows for anti-metastatic target identification and intravital analysis of a dynamic and complex cascade of events.

This is an effective method to screen for suppressors or drivers of cancer metastasis. Cells, transduced with an expression library, are injected into the chicken chorioallantoic membrane vasculature to form metastatic colonies. Colonies having decreased or increased invasiveness are excised, expanded, reinjected to confirm their phenotype, and finally, analyzed using high throughput sequencing.

Recent advances in cancer research has illustrated the highly complex nature of cancer metastasis. Multiple genes or genes networks have been found to be ...

5-Aminolevulinic Acid-mediated Photodynamic Therapy on Primary Bone Tumor and Bone Metastases Cell Lines

Bone metastases are associated with poor prognosis and low quality of life for the affected patients. Photodynamic therapy (PDT) emerges as a noninvasive therapy that can target local metastatic bone lesions. This paper presents an in vitro method to study the PDT effect in adherent cell lines. To this end, we demonstrate a step-by-step approach to subject both primary (giant cell bone tumor) and human bone metastatic cancer cell lines (derived from a primary invasive ductal breast carcinoma and renal carcinoma) to 5-aminolevulinic acid (5-ALA)-mediated PDT. 
After 24 h post 5-ALA-PDT irradiation (blue light-wavelength 436 nm), the therapeutic effect was assessed in terms of cell migration potential, viability, apoptotic features, and cellular growth arrest (senescence). Post 5-ALA-PDT irradiation, musculoskeletal-derived cell lines respond differently to the same doses and exposure of PDT. Depending on the extent of cellular damage triggered by PDT exposure, two different cell fates-apoptosis and senescence were noted. Variable sensitivity to PDT therapy among different bone cancer cell lines provides useful information for selecting more appropriate PDT settings in clinical settings. This protocol is designed to exemplify the use of PDT in the context of musculoskeletal neoplastic cell lines. It may be adjusted to investigate the therapeutic effect of PDT on various cancer cell lines and various photosensitizers and light sources.

This protocol presents a step-by-step methodological approach of exposing bone metastatic cells lines and primary bone tumors to 5-aminolevulinic acid-mediated photodynamic therapy (PDT). The effects on cell migration potential/invasiveness, viability, apoptosis, and senescence potential are also analyzed following PDT exposure.

Bone metastases are associated with poor prognosis and low quality of life for the affected patients. Photodynamic therapy (PDT) emerges as a noninvasive ...

Cytotoxic Efficacy of Photodynamic Therapy in Osteosarcoma Cells In Vitro

In recent years, there has been the difficulty in finding more effective therapies against cancer with less systemic side effects. Therefore Photodynamic Therapy is a novel approach for a more tumor selective treatment.
Photodynamic Therapy (PDT) that makes use of a nontoxic photosensitizer (PS), which, upon activation with light of a specific wavelength in the presence of oxygen, generates oxygen radicals that elicit a cytotoxic response1. Despite its approval almost twenty years ago by the FDA, PDT is nowadays only used to treat a limited number of cancer types (skin, bladder) and nononcological diseases (psoriasis, actinic keratosis)2.
The major advantage of the use of PDT is the ability to perform a local treatment, which prevents systemic side effects. Moreover, it allows the treatment of tumors at delicate sites (e.g. around nerves or blood vessels). Here, an intraoperative application of PDT is considered in osteosarcoma (OS), a tumor of the bone, to target primary tumor satellites left behind in tumor surrounding tissue after surgical tumor resection. The treatment aims at decreasing the number of recurrences and at reducing the risk for (postoperative) metastasis.
In the present study, we present in vitro PDT procedures to establish the optimal PDT settings for effective treatment of widely used OS cell lines that are used to reproduce the human disease in well established intratibial OS mouse models. The uptake of the PS mTHPC was examined with a spectrophotometer and phototoxicity was provoked with laser light excitation of mTHPC at 652 nm to induce cell death assessed with a WST-1 assay and by the counting of surviving cells. The established techniques enable us to define the optimal PDT settings for future studies in animal models. They are an easy and quick tool for the evaluation of the efficacy of PDT in vitro before an application in vivo.

Cytotoxic Efficacy of Photodynamic Therapy in Osteosarcoma Cells In Vitro

The protocol reported here allows the evaluation of the efficacy of photodynamic therapy (PDT) in cell lines and the optimization of the PDT settings before applying the therapy in animal models.

In recent years, there has been the difficulty in finding more effective therapies against cancer with less systemic side effects. Therefore Photodynamic ...

Medicine

Implantation and Evaluation of Melanoma in the Murine Choroid via Optical Coherence Tomography

Establishing experimental choroidal melanoma models is challenging in terms of the ability to induce tumors at the correct localization. In addition, difficulties in observing posterior choroidal melanoma in vivo limit tumor location and growth evaluation in real-time. The approach described here optimizes techniques for establishing choroidal melanoma in mice via a multi-step sub-choroidal B16LS9 cell injection procedure. To enable precision in injecting into the small dimensions of the mouse uvea, the complete procedure is performed under a microscope. First, a conjunctival peritomy is formed in the dorsal-temporal area of the eye. Then, a tract into the sub-choroidal space is created by inserting a needle through the exposed sclera. This is followed by the insertion of a blunt needle into the tract and the injection of melanoma cells into the choroid. Immediately after injection, noninvasive optical coherence tomography (OCT) imaging is utilized to determine tumor location and progress. Retinal detachment is evaluated as a predictor of tumor site and size. The presented method enables the reproducible induction of choroid-localized melanoma in mice and the live imaging of tumor growth evaluation. As such, it provides a valuable tool for studying intraocular tumors.

Implantation and Evaluation of Melanoma in the Murine Choroid via Optical Coherence Tomography

The present protocol describes the implantation and evaluation of melanoma in the murine choroid utilizing optical coherence tomography.

Establishing experimental choroidal melanoma models is challenging in terms of the ability to induce tumors at the correct localization. In addition, ...

An In-House-Built and Light-Emitting-Diode-Based Photodynamic Therapy Device for Enhancing Verteporfin Cytotoxicity in a 2D Cell Culture Model

This paper describes a novel, simple, and low-cost device to perform in vitro photodynamic therapy (PDT) assays, named the PhotoACT. The device was built using a set of conventional programmable light-emitting diodes (LEDs), a liquid crystal display (LCD) module, and a light sensor connected to a commercial microcontroller board. The box-based structure of the prototype was made with medium-density fiberboards (MDFs). The internal compartment can simultaneously allocate four cell culture multiwell microplates.
As a proof of concept, we studied the cytotoxic effect of the photosensitizer (PS) verteporfin against the HeLa cell line in two-dimensional (2D) culture. HeLa cells were treated with increasing concentrations of verteporfin for 24 h. The drug-containing supernatant medium was discarded, the adherent cells were washed with phosphate-buffered saline (PBS), and drug-free medium was added. In this study, the effect of verteporfin on cells was examined either without light exposure or after exposure for 1 h to light using red-green-blue (RGB) values of 255, 255, and 255 (average fluence of 49.1 &#177; 0.6 J/cm2). After 24 h, the cell viability was assessed by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) assay.
Experimental results showed that exposure of cells treated with verteporfin to the light from the device enhances the drug&#39;s cytotoxic effect via a mechanism mediated by reactive oxygen species (ROS). In addition, the use of the prototype described in this work was validated by comparing the results with a commercial PDT device. Thus, this LED-based photodynamic therapy prototype represents a good alternative for in vitro studies of PDT.

Here, we describe a novel, simple, and low-cost device to successfully perform in vitro photodynamic therapy (PDT) assays using two-dimensional HeLa cell culture and verteporfin as a photosensitizer.

This paper describes a novel, simple, and low-cost device to perform in vitro photodynamic therapy (PDT) assays, named the PhotoACT. The device was built ...

Biochemistry

Pulsed Laser Diode-Based Desktop Photoacoustic Tomography for Monitoring Wash-In and Wash-Out of Dye in Rat Cortical Vasculature

Photoacoustic (PA) tomography (PAT) imaging is an emerging biomedical imaging modality useful in various preclinical and clinical applications. Custom-made circular ring array-based transducers and conventional bulky Nd:YAG/OPO lasers inhibit translation of the PAT system to clinics. Ultra-compact pulsed laser diodes (PLDs) are currently being used as an alternative source of near-infrared excitation for PA imaging. High-speed dynamic in vivo imaging has been demonstrated using a compact PLD-based desktop PAT system (PLD-PAT). A visualized experimental protocol using the desktop PLD-PAT system is provided in this work for dynamic in vivo brain imaging. The protocol describes the desktop PLD-PAT system configuration, preparation of animal for brain vascular imaging, and procedure for dynamic visualization of indocyanine green (ICG) dye uptake and clearance process in rat cortical vasculature.&#160;

A compact pulsed laser diode-based desktop photoacoustic tomography (PLD-PAT) system is demonstrated for high-speed dynamic in vivo imaging of small animal cortical vasculature.

Photoacoustic (PA) tomography (PAT) imaging is an emerging biomedical imaging modality useful in various preclinical and clinical applications. ...

Bioengineering

An In Vitro Approach to Photodynamic Therapy

Photodynamic therapy (PDT) is a medical procedure that involves incubation of an exogenously applied photosensitizer (PS) followed by visible light photoactivation to induce cell apoptosis. The Federal Drug Administration has approved PDT for the treatment of actinic keratosis, and clinical guidelines recommend PDT as a treatment for certain non-melanoma skin cancers and acne vulgaris. PDT is an advantageous therapeutic modality as it is low cost, non-invasive, and associated with minimal adverse events and scaring. In the first step of PDT, a PS is applied and allowed to accumulate intracellularly. Subsequent light irradiation induces reactive oxygen species formation, which may ultimately lead to cell apoptosis, membrane disruption, mitochondrial damage, immune modulation, keratinocyte proliferation, and collagen turnover. Herein, we present an in vitro method to study PDT in an adherent cell line. This treatment protocol is designed to simulate PDT and may be adjusted to studying the use of PDT with various cell lines, photosensitizers, incubation temperatures, or photoactivation wavelengths. Squamous cell carcinoma cells were incubated with 0, 0.5, 1.0, and 2 mM 5-aminolevulinic acid (5-ALA) for 30 min and photoactivated with 417 nm blue light for 1,000 s. The primary outcome measure was apoptosis and necrosis, as measured by annexin-V and 7-aminoactinomycin D flow cytometry. There was a dose-dependent increase in cell apoptosis following thirty-minute incubation of 5-ALA. To achieve high inter-test validity, it is important to maintain consistent incubation and light parameters when performing in vitro PDT experiments. PDT is a useful clinical procedure and in vitro research may allow for the development of novel PSs, optimization of protocols, and new indications for PDT.

An In Vitro Approach to Photodynamic Therapy

Photodynamic therapy (PDT) is a medical procedure that involves incubation of an exogenously applied photosensitizer (PS) followed by visible light photoactivation to induce apoptosis. We present an in vitro PDT protocol designed to simulate PDT that may be used to study differences in PS incubation and light treatment parameters.

Photodynamic therapy (PDT) is a medical procedure that involves incubation of an exogenously applied photosensitizer (PS) followed by visible light ...

Chick Chorioallantoic Membrane Harvest: A Method to Isolate Tumor Graft-bearing Chorioallantoic Membrane from Chicken Egg

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)
This video demonstrates the isolation of tumor-bearing chorioallantoic membrane from the chicken embryo. The harvested CAM can be used for further downstream applications.

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)
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Quail Chorioallantoic Membrane - A Tool for Photodynamic Diagnosis and Therapy

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An In Vitro Approach to Photodynamic Therapy

Chick Chorioallantoic Membrane Harvest: A Method to Isolate Tumor Graft-bearing Chorioallantoic Membrane from Chicken Egg